Method of using eukaryotic expression vectors comprising the BK virus enhancer

ABSTRACT

The present invention is a method of using the BK enhancer in tandem with a eukaryotic promoter to promote transcription of DNA that encodes a useful substance. The method of the present invention requires the presence of the E1A gene product for maximum expression of the useful substance. The present invention also comprises a number of useful expression vectors that comprise the BK enhancer in tandem with the adenovirus 2 late promoter positioned to drive expression of a variety of proteins, such as protein C, chloramphenicol acetyltransferase, and tissue plasminogen activator. The present invention further comprises a method for increasing the activity of the BK enhancer involving placement of the BK enhancer immediately upstream of the eukaryotic promoter used in tandem with the BK enhancer to drive expression of a useful substance. Furthermore, the present invention also comprises a method for coamplification of genes in primate cells. Additionally, the invention further comprises the recombinant human protein C molecule produced in 293 cells which comprises novel glycosylation patterns.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division, of application Ser. No. 08/208,930 filedMar. 9, 1994, now U.S. Pat. No. 5,550,036, which is a continuation ofapplication Ser. No. 07/368,700, filed Jun. 20, 1989, now abandoned,which is a continuation in part of application Ser. No. 07/250,001,filed Sep. 27, 1988, now abandoned, which is a continuation in part ofapplication Ser. No. 07/129,028, filed Dec. 4, 1987, now abandoned,which is a continuation in part of application Ser. No. 849,999, filedApr. 9, 1986, now abandoned.

BACKGROUND OF THE INVENTION

The present invention concerns a method of using the BK enhancer in thepresence of an immediate-early gene product of a large DNA virus inincrease transcription of a recombinant gene in eukaryotic host cells.The BK enhancer is a defined segment of DNA that consists of threerepeated sequences (the prototye BK enhancer is depicted in Example 17,below). However, a wide variety of BK enhancer variants, not allconsisting of three repeated sequences, are known in the art andsuitable for use in the invention.

The BK enhancer sequence exemplified herein is obtained from BK virus, ahuman papovavirus that was first isolated from the urine of animmunosuppressed patient. BK virus is suspected of causing an unapparentchildhood infection and is ubiquitous in the human population. AlthoughBK virus grows optimally in human cells, the virus undergoes an abortivecycle in non-primate cells, transforms rodent cells in vitro, andinduces tumors in hamsters. BK virus is very similar to SV40, but theenhancer sequences of the two papovaviruses, SV40 and BK, differsubstantially in nucleotide sequence. The complete nucleotide sequenceof BK virus (˜5.2 kb) has been disclosed by Seif et al., 1979, Cell18:963, and Yang and Wu, 1979, Science 206:456. Prototype BK virus isavailable from the American Type Culture Collection (ATCC), 12301Parklawn Dr., Rockville, Md. 20852-1776, under the accession number ATCCVR-837. A restriction site and function map of prototype BK virus ispresented in FIG. 1 of the accompanying drawings.

Enhancer elements are cis-acting and increase the level of transcriptionof an adjacent gene from its promoter in a fashion that is relativelyindependent of the position and orientation of the enhancer element. Infact, Khoury and Gruss, 1983, Cell 33:313, state that “the remarkableability of enhancer sequences to function upstream from, within, ordownstream from eukaryotic genes distinguishes them from classicalpromoter elements . . . ” and suggest that certain experimental resultsindicate that “enhancers can act over considerable distances(perhaps >10 kb).”

The present invention teaches that unexpected increases in transcriptionresult upon positioning the BK enhancer immediately upstream of (on the5′ side of) the “CAAT” region of a eukaryotic promoter that is used intandem with the BK enhancer to transcribe a DNA sequence encoding auseful substance. The CAAT region or “immediate upstream region” or “-80homology sequence” is a cis-acting upstream element that is a conservedregion of nucleotides observed in promoters whose sequences fortranscriptional activity have been dissected. The CAAT region is foundin many, but not all, promoters. In other promoters, equivalentcis-acting upstream elements are found, including SP1 binding sites, theocta sequence, nuclear factor 1 binding sits, the AP1 and AP2homologies, glucocorticoid response elements, and heat shock responseelements. The CAAT region equivalent in the adenovirus major latepromoter is the upstream transcription factor (UTF) binding site(approximate nucleotides −50 to −65 upstream of the CAP site). The CAATsequence mediates the efficiency of transcription and, with fewexceptions, cannot be deleted without decreasing promoter strength.

Enhancer elements have been identified in a number of viruses, includingpolyoma virus, papiloma virus, adenovirus, retrovirus, hepatitis virus,cytomegalovirus, herpes virus, papovaviruses, such as simian virus 40(SV40) and BK, and in many non-viral genes, such as within mouseimmunoglobulin gene introns. Enhancer elements may also be present in awide variety of other organisms. Host cells often react differently todifferent enhancer elements. This cellular specificity indicates thathost gene products interact with the enhancer element during geneexpression.

Enhancer elements can also interact with viral gene products present inthe host cell. Velcich and Ziff, 1983, Cell 40:705; Borrelli et al.,1984, Nature 312:608; and Hen et al., 1985, Science 230:1391, disclosethat the adenovirus-2 early region 1A (E1A) gene products repressactivation of transcription induced by the SV40, polyoma virus, mouseimmunoglobulin gene and adenovirus-2 E1A enhancers. Eukaryoticexpression vectors that utilized enhancers to increase transcription ofrecombinant genes consequently were not expected to work better thanvectors without enhancers in E1A-containing host cells. In strikingcontrast to the prior art methods of using enhancers, the present methodfor using the BK virus enhancer element involves using the E1A geneproduct or a similar immediate-early gene product of a large DNA virusto maximize gene expression. Thus, the present invention teaches thatthe ability of the BK enhancer to promoter transcription of DNA isincreased in the presence of the E1A gene product of any adenovirus.

The E1A gene product (actually, the E1A gene produces two products,which are collectively referred to herein as “the E1A gene product”) isan immediate-early gene product of adenovirus, a large DNA virus. Thepresent invention encompasses the use of any immediate-early geneproduct of a large DNA virus that functions similarly to the E1A geneproduct to increase the activity of the BK enhancer. The herpes simplexvirus ICP4 protein, described by DeLuca et al., 1985, Mol. Cell. Biol.5: 1997-2008, the pseudorabies virus IE protein, described by Feldman etal., 1982 P.N.A.S. 79:4952-4956, and the E1B protein of adenovirus areall immediate-early gene products of large DNA viruses that havefunctions similar to the E1A protein. Therefore, the method of thepresent invention includes the use of the ICP4, IE, or E1B proteins,either in the presence or absence of E1A protein, to increase theactivity of the BK enhancer.

SUMMARY OF THE INVENTION

The present invention concerns a method of using the BK virus enhancerin the presence of an immediate-early gene product of a large DNA virus,such as the EIA gene product of adenovirus, for purposes of increasingtranscription and expression of recombinant genes in eukaryotic hostcells. Another significant aspect of the present invention relates to avariety of expression vectors that utilize the BK enhancer sequence intandem with a eukaryotic promoter, such as the adenovirus late promoter(MLP), to drive expression of useful products in eukaryotic host cells.Many of these expression vectors comprise a BK enhancer-adenovirus latepromoter cassette, which can be readily transferred to other vectors foruse in the present method. The versatility of the present expressionvectors is demonstrated by the high-level expression driven by thesevectors of such diverse proteins as chloramphenicol acetyltransferase,protein C, tissue plasminogen activator, and modified tissue plasminogenactivator.

In the construction of certain vectors of the invention, the BK enhancerand SV40 enhancer were placed in tandem at the front (5′) end of theMLP, itself positioned to drive expression of a recombinant gene on arecombinant DNA expression vector. This tandem placement yieldedunexpectedly higher levels of expression in cells that did not expressthe immediate-early gene product of a large DNA virus. Consequently, afurther aspect of the invention is a method of producing a gene productin a recombinant host cell that comprises transforming the host cellwith a recombinant DNA vector that comprises two different enhancersplaced at the 5′ end of the coding sequence for the gene product andculturing the transformed cell under conditions that allow for geneexpression.

The practice of the invention to express human protein C inadenovirus-transformed cells led to the discovery that such cells areespecially preferred hosts for the production of γ-carboxylatedproteins. Consequently, a further aspect of the invention comprises amethod for making γ-carboxylated proteins.

Yet another important aspect of the present invention concerns a methodof increasing the activity of the BK enhancer relative to an adjacenteukaryotic promoter and is illustrated using the BKenhancer-adenovirus-2 late promoter cassette. These derivatives wereconstructed by enzymatic treatment that positioned the BK enhancer veryclose to the CAAT region of the adenovirus-2 late promoter. Dramaticincreases in expression levels, as compared with constructions that lackthis positioning, were observed when these modified BKenhancer-adenovirus late promoter sequences were incorporated intoexpression vectors and then used to drive expression of useful geneproducts in eukaryotic host cells. Thus, the present invention providesa method for increasing the activity of the BK enhancer relative to anadjacent eukaryotic promoter that comprises positioning the enhancerimmediately upstream, within 0 to about 300 nucleotides, of the 5′ endof the CAAT region or CAAT region equivalent of the eukaryotic promoter.

Yet another aspect of the invention results from attempts to increaseexpression of recombinant products encoded on the vectors describedherein by incorporation of portions of the tripartite leader sequence ofadenovirus into those expression vectors. Significant increases inexpression result when the first part of the tripartite leader ofadenovirus is encoded into a recombinant DNA expression vector, and suchexpression can be further increased in some situations by action of theVA gene product of adenovirus.

An additional aspect of the present invention concerns a method ofamplification of genes in primate cells. The most widely used method forgene amplification employs the murine dihydrofolate reductase gene forselection and amplification in a dhfr deficient cell line. Humanpolypeptides often require post-translational modifications which occurmost efficiently in primate cells, yet most primate cells cannot bedirectly selected or amplified using only the dhfr system. The presentinvention provides a method wherein the primate cells are first isolatedusing a directly selectable marker, then amplified using the dhfrsystem, thereby significantly increasing the expression levels fromprimate cells.

Another aspect of the present invention concerns novel recombinantlyproduced human protein C molecules which contain glycosylation patternstotally unlike the human protein C molecules derived from plasma. Thenovel recombinantly produced protein C molecules display functionalactivities which are quite different than plasma-derived human proteinC. Furthermore, the recombinant human protein C molecules derived from293 cells contain fewer sialic acid residues than the plasma-derivedhuman protein C.

For purposes of the present invention, the following terms are asdefined below.

Antibiotic—a substance produced by a microorganism that, eithernaturally or with limited chemical modification, will inhibit the growthof or kill another microorganism or eukaryotic cell.

Antibiotic Resistance-Conferring Gene—a DNA segment that encodes anactivity that confers resistance to an antibiotic.

ApR—the ampicillin-resistant phenotype or gene conferring same.

Cloning—the process of incorporating a segment of DNA into a recombinantDNA cloning vector.

CmR—the chloramphenicol-resistant phenotype or gene conferring same.

dhfr—dihydrofolate reductase.

ep—a DNA segment comprising the SV40 early promoter of the T-antigengene, the T-antigen binding sites, and the SV40 origin of replication.

Eukaryotic promoter—any DNA sequence that functions as a promoter ineukaryotic cells.

HmR—the hygromycin-resistant phenotype or gene conferring same.

IVS—DNA encoding an intron, also called an intervening sequence.

Large DNA virus—a virus that infects eukaryotic cells and has a genomegreater than ˜10 kb in size, i.e., any of the pox viruses, adenoviruses,and herpes viruses.

MLP—the major late promoter of adenovirus, which is also referred toherein as the late promoter of adenovirus.

NeoR—the neomycin resistance-conferring gene, which can also be used toconfer G418 resistance in eukaryotic host cells.

ori—a plasmid origin of replication.

pA—a DNA sequence encoding a polyadenylation signal.

Promoter—a DNA sequence that directs transcription of DNA into RNA.

Recombinant DNA Cloning Vector—any autonomously replicating orintegrating agent that comprises a DNA molecule to which one or moreadditional DNA segments can be or have been added.

Recombinant DNA Expression Vector—any recombinant DNA cloning vectorcomprising a promoter and associated insertion site, into which a DNAmolecule that encodes a useful product can be inserted and expressed.

Recombinant DNA Vector—any recombinant DNA cloning or expression vector.

Replicon—any DNA sequence that controls the replication of a recombinantDNA vector.

Restriction Fragment—any linear DNA generated by the action of one ormore restriction enzymes.

rRNA—ribosomal ribonucleic acid.

Sensitive Host Cell—a host cell that cannot grow in the presence of agiven antibiotic or other toxic compound without a DNA segment thatconfers resistance thereto.

Structural Gene—any DNA sequence that encodes a polypeptide, inclusiveof that DNA encoding the start and stop codons.

Structural Polypeptide—any useful polypeptide, including, but notlimited to, human protein C, tissue plasminogen activator, insulin,thrombomodulin, factor Va or factor VIIIa.

TcR—the tetracycline-resistant phenotype or gene conferring same.

Transformant—a recipient host cell that has undergone transformation.

Transformation—the introduction of DNA into a recipient host cell.

tRNA—transfer ribonucleic acid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a restriction site and function map of BK virus.

FIG. 2 is a restriction site and function map of plasmid pBKE1.

FIG. 3 is a restriction site and function map of plasmid pBKneo1.

FIG. 4 is a restriction site and function map of plasmid pSV2cat.

FIG. 5 is a restriction site and function map of plasmid pLPcat.

FIG. 6 is a restriction site and function map of plasmid pBLcat.

FIG. 7 is a restriction site and function map of plasmid pBKcat.

FIG. 8 is a restriction site and function map of plasmid pSBLcat.

FIG. 9 depicts the construction and presents a restriction site andfunction map of plasmid pL133.

FIG. 10 is a restriction site and function map of plasmid pLPC.

FIG. 11 is a restriction site and function map of plasmid pLPC4.

FIG. 12 is a restriction site and function map of plasmid pSV2hyg.

FIG. 13 is a restriction site and function map of plasmid pLPChyg1.

FIGS. 14A-14P depict the construction and presents a restriction siteand function map of plasmid pBW32.

FIG. 15 is a restriction site and function map of plasmid pLPChd1.

FIG. 16 is a restriction site and function map of plasmid phd.

FIG. 17 is a restriction site and function map of plasmid pLPCE1A.

FIG. 18 is a restriction site and function map of plasmid pBLT.

FIG. 19 is a restriction site and function map of plasmid pBLThyg1.

FIG. 20 is a restriction site and function map of plasmid pBLTdhfr1.

FIG. 21 is a restriction site and function map of plasmid pTPA602.

FIG. 22 is a restriction site and function map of plasmid pTPA603.

FIG. 23 is a restriction site and function map of plasmid phdTPA.

FIG. 24 is a restriction site and function map of plasmid phdMTPA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns an improved method for producing a usefulsubstance in a eukaryotic host cell wherein said cell is transformedwith a recombinant DNA vector that comprises a eukaryotic promoter, a BKenhancer positioned to stimulate said promoter, and a DNA sequence thatencodes said useful substance, said sequence being positioned forexpression from said promoter, and wherein said cell containing saidvector is cultured under conditions suitable for expression of saiduseful substance, wherein the improvement comprises: (a) providing saidcell with a DNA sequence that codes for the expression of animmediate-early gene product of a large DNA virus; and (b) culturingsaid cell of step a) under conditions suitable for expressing said geneproduct and stimulating the activity of said enhancer. Those skilled inthe art recognize that many established cell lines express animmediate-early gene product of a large DNA virus and that such celllines are especially useful in the present method. Thus, the presentinvention also comprises an improved method for producing a usefulsubstance in a eukaryotic host cell wherein said cell is transformedwith a recombinant DNA vector that comprises a eukaryotic promoter, a BKenhancer positioned to stimulate said promoter, and a DNA sequence thatencodes said useful substance, said sequence being positioned forexpression from said promoter, and wherein said cell containing saidvector is cultured under conditions suitable for expression of saiduseful substance, wherein the improvement comprises: (a) inserting saidvector into a eukaryotic host cell that expresses an immediate-earlygene product of a large DNA virus, and (b) culturing said cell of stepa) under conditions suitable for expressing said gene product andstimulating the activity of said enhancer.

An important aspect of the present invention is the novel group ofexpression vectors that comprise the BK enhancer sequence in tandem withthe adenovirus-2 late promoter. The expression vectors of the presentinvention were constructed so that DNA molecules encoding usefulproducts can be or have been readily inserted into the vectors in thecorrect position for expression. Furthermore, the BK enhancer sequenceand eukaryotic promoter have been constructed to form a “cassette,”which can be isolated from the expression vectors on a relatively smallrestriction fragment. The cassette can be readily shuttled between avariety of expression vectors. The expression vectors specificallyexemplified herein utilize the adenovirus-2 or BK late promoter in theBK enhancer-eukaryotic promoter cassette that drives transcription inthe method of the present invention.

Although BK virus (ATCC VR-837) can be purchased or readily isolated inlarge quantities as described in Example 1, it is also convenient toclone the BK viral DNA onto a plasmid cloning vector and use therecombinant vector as a source of BK viral DNA sequences. Consequently,BK viral DNA was digested with restriction enzyme EcoRI, which, due tothe presence of only one EcoRI site on the BK genome, produced linear BKDNA. Plasmid pUC8 (available from Bethesda Research Laboratories (BRL),P.O. Box 6009, Gaithersburg, Md. 20877) was likewise digested andlinearized with restriction enzyme EcoRI, and the EcoRI-cut plasmid pUC8DNA was ligated to the EcoRI-cut BK viral DNA to form plasmids pBKE1 andpBKE2, which differ only with respect to the orientation of the BK viralDNA. A restriction site and function map of plasmid pBKE1 is presentedin FIG. 2 of the accompanying drawings. The construction of plasmidspBKE1 and pBKE2 is described in Example 2.

The BK viral genome has also been combined with a portion of plasmidpdBPV-MMTneo to construct plasmids pBKneo1 and pBKneo2. PlasmidpdBPV-MMTneo, about 15 kb in size and available from the ATCC under theaccession number ATCC 37224, comprises the replicon and p-lactamase genefrom plasmid pBR322, the mouse metal-lothionein promoter positioned todrive expression of a structural gene that encodes a neomycinresistance-conferring enzyme, and about 8 kb of bovine papilloma virus(BPV) DNA. Plasmid pdBPV-MMTneo can be digested with restriction enzymeBamHI to generate two fragments: the ˜8 kb fragment that comprises theBPV DNA and an ˜7 kb fragment that comprises the other sequencesdescribed above. BK virus has only one BamHI restriction site, andplasmids pBKneo1 and pBKneo2 were constructed by ligating the ˜7 kbBamHI restriction fragment of plasmid pdBPV-MMTneo to BamHI-linearizedBK virus DNA. The construction of plasmids pBKneo1 and pBKneo2, whichdiffer only with respect to the orientation of the BK virus DNA, isdescribed in Example 3, and a restriction site and function map ofplasmid pBKneo1 is presented in FIG. 3 of the accompanying drawings.

Plasmids pBKE1, pBKE2, pBKneo1, and pBKneo2 each comprise the entiregenome of the BK virus, including the enhancer sequence, and thus serveas useful starting materials for the expression vectors of the presentinvention. One such illustrative expression vector, plasmid pBLcat,comprises the BK enhancer sequence in tandem with the humanadenovirus-type-2 late promoter positioned to drive expression of thechloramphenicol acetyltransferase enzyme (CAT). Plasmid pSV2cat servesas a convenient source of the CAT gene and can be obtained from the ATCCunder the accession number ATCC 37155. A restriction site and functionmap of plasmid pSV2cat is presented in FIG. 4 of the accompanyingdrawings. Human adenovirus-type-2 DNA is commercially available and canalso be obtained from the ATCC under the accession number ATCC VR-2.

Illustrative plasmid pBLcat was constructed by ligating the ˜0.32 kblate-promoter-containing AccI-PvuII restriction fragment of humanadenovirus-type-2 DNA to blunt-ended BclI linkers that attached only tothe PvuII end of the AccI-PvuII restriction fragment. The resultingfragment was then ligated to the ˜4.51 kb AccI-StuI restriction fragmentof plasmid pSV2cat to yield intermediate plasmid pLPcat, for which arestriction site and function map is presented in FIG. 5 of theaccompanying drawings. The desired plasmid pBLcat was constructed fromplasmid pLPcat by ligating the origin of replication andenhancer-containing, ˜1.28 kb AccI-PvuII restriction fragment of BKvirus DNA to the ˜4.81 kb AccI-StuI restriction fragment of plasmidpLPcat. A restriction site and function map of the resulting plasmidpBLcat is presented in FIG. 6 of the accompanying drawings. Theconstruction of plasmid pBLcat is further described in Example 4.

Plasmid pBKcat is an expression vector that further exemplifies thepresent invention and utilizes the BK enhancer and BK late promoter todrive expression of chloramphenicol acetyltransferase. Plasmid pBKcatwas constructed in a manner analogous to that described for plasmidpLPcat. Thus, the ˜4.51 kb AccI-StuI restriction fragment of plasmidpSV2cat was ligated to the ˜1.28 kb AccI-PvuII restriction fragment ofBK virus such that the BK late promoter is in the correct orientation todrive expression of the CAT gene. A restriction site and function map ofplasmid pBKcat is presented in FIG. 7 of the accompanying drawings.

Plasmid pBLcat is a convenient source of the BK enhancer-adenovirus latepromoter “cassette” of the present invention. This cassette is an ˜870bp HindIII restriction fragment that can be conveniently inserted into aeukaryotic expression vector to increase expression of a product encodedby that vector. This was done by digesting plasmid pSv2cat withrestriction enzyme HindIII and inserting the BK enhancer-adenovirus latepromoter cassette. The resultant plasmid, designated as plasmid pSBLcat,contains the SV40 origin of replication, SV40 early promoter, and SV40enhancer and therefore differs from plasmid pBLcat in which thosesequences have been deleted. The tandem SV40 enhancer-BKenhancer-adenovirus major late promoter (SBL promoter) cassette can beexcised from plasmid pSBLcat on a PvuII restriction enzyme fragment,which can be conveniently inserted into any recombinant DNA expressionvector.

Plasmid pSBLcat drives expression of CAT to higher levels than doesplasmid pBLcat, so long as no E1A gene product is present. Thisincreased expression in the absence of E1A gene product indicates thatthe two enhancers, one from SV40 and the other from BK, have anadditive, enhancing effect on transcription from nearby promoters. Toassess the strength and utility of the SBL promoter, the chloramphenicolacetyltransferase (CAT) expression vector, pSBL-CAT, was transfectedvector into a variety of mammalian host cells, and the level of CATactivity was measured 48 to 72 hours later as described by Gorman, etal., 1982, Mol. Cell. Biol. 2:1044-1051. The level of CAT activityobtained from pSV2-CAT, in which the CAT gene is driven by the strongSV40 early promoter, was used for comparative purposes. The SBL promoterwas 3 to 6 fold stronger than the SV40 early promoter in the followingcell lines: BHK-21, HeLa; MK2, COS-1, 293, CHO (all available from theAmerican Type Culture collection), P3UCLA (Varki et al., 1984, CancerRes. 44:681-687), K816 (Grinnell et al., 1986, Mol. Cell. Biol.6:3596-3605), and an adenovirus-transformed Syrian hamster tumor line,AV12, described below. In primary human embryonic kidney cells and livercells, CAT activity was detected after transfection with PSBL-CAT, butnot with pSV2-CAT. Although efficient expression from the MLP could beobtained with either the BK (pBL-CAT) or SV40 enhancer (pSL-CAT, aplasmid that is analogous to plasmid pSV2-CAT, except that the SV40early promoter is replaced with the adenovirus 2 major late promoter,described by Grinnell et al., 1986, Mol. Cell. Biol. 6:3596-3605), thesesingle enhancer constructions did not function efficiently in all cells.For example, pSV2-CAT was 3 fold stronger than pSL-CAT in 293 cells and10 fold stronger than pBL-CAT in HeLa cells. Thus, the use of tandemenhancer sequences upstream of a eukaryotic promoter results in a strongand versatile promoter that displays little host cell dependence, andtherefore can be used for the efficient expression of genes in a widevariety of mammalian cells.

However, in the presence of E1A gene product, plasmid pBLcat drivesexpression of CAT to higher levels than does plasmid pSBLcat, presumablybecause the SV40 enhancer is inhibited by the E1A gene product.Conversely, in HeLa cells, the SV40 enhancer stimulated transcriptionfrom the adenovirus 2 major late promoter (Ad2MLP) 26 fold, but the BKenhancer only stimulated transcription from Ad2MLP 1.5 fold in HeLacells. Because the basal level of BK activity in HeLa cells is so low,stimulation of that activity with the immediate-early gene product of alarge DNA virus, such as E1A protein, still does not result in optimalexpression levels. This low level activity of the BK enhancer in HeLacells is thought to be due to a repressor activity present in HeLa cellsthat interacts with the BK enhancer. This repressor activity in HeLacells can be titrated out by introducing more copies of the BK enhancerinto the HeLa cell. In fact, in the HeLa cell line, E1A may increase thelevel of the repressor. However, optimal expression levels can beobtained in HeLa cells using the tandem SV40 enhancer BK enhancer of theinvention. This tandem enhancer thus has the advantage of avoidingcell-specific negative interactions that may be encountered, as in HeLacells, in some host cells. A restriction site and function map ofplasmid pSBLcat is presented in FIG. 8 of the accompanying drawings, andthe construction of plasmid pSBLcat is described in Example 5.

The BK enhancer-adenovirus late promoter cassette has also been used toimprove expression of human protein C. This was done by ligating thecassette into plasmid pL133, a plasmid disclosed and claimed in U.S.patent application Ser. No. 699,967, filed Feb. 8, 1985, incorporatedherein by reference. A restriction site and function map of plasmidpL133 is presented in FIG. 9 of the accompanying drawings. PlasmidpL133, the construction of which is given in Example 6, was digestedwith restriction enzyme HindIII and then ligated to the ˜0.87 kb HindIIIrestriction, fragment of plasmid pBLcat to yield plasmid pLPC. Arestriction site and function map of plasmid pLPC is presented in FIG.10 of the accompanying drawings, and the construction of plasmid pLPC isfurther described in Example 7.

Plasmid pLPC, like plasmid PL133, comprises the enhancer, early and latepromoters, T-antigen-binding sites, and origin of replication of SV40.Thus, use of plasmid pLPC and derivatives thereof in any recombinanthost cells is illustrative of the tandem enhancer expression method ofthe invention. Plasmid pLPC served as a useful starting material formany vectors of the invention, including plasmid pSBL. Plasmid pSBL wasconstructed by deleting the protein C-encoding DNA on plasmid pLPC. Thisdeletion merely requires excision of plasmid pLPC's single BclIrestriction fragment by digestion with BclI and self-ligation. Theresulting plasmid pSBL serves as a convenient expression vector for usein the tandem enhancer method of the invention, for coding sequences ofinterest can be readily inserted at the sole remaining BclI site.

The SV40 elements present on plasmid pLPC are situated closely togetherand difficult to delineate. The binding of T antigen to theT-antigen-binding sites, which is necessary for SV40 replication, isknown to enhance transcription from the SV40 late promoter andsurprisingly has a similar effect on the BK later promoter. Because thehigh level of T-antigen-driven replication of a plasmid that comprisesthe SV40 origin of replication is generally lethal to the host cell,neither plasmid pLPC nor plasmid pL133 are stably maintained as episomal(extrachromosomal) elements in the presence of SV40 T antigen, butrather, the two plasmids must integrate into the chromosomal DNA of thehost cell to be stably maintained.

The overall structure of the BK enhancer region is quite similar to thatof SV40, for the BK enhancer, origin of replication, early and latepromoters, and the BK analogue of the T-antigen-binding sites are allclosely situated and difficult to delineate on the BK viral DNA.However, when grown in the presence of BK T antigen, a plasmid thatcomprises the BK origin of replication and T-antigen-binding sites doesnot replicate to an extent that proves lethal and is stably maintainedas an episomal element in the host cell. In addition, theT-antigen-driven replication can be used to increase the copy number ofa vector comprising the BK origin of replication so that when selectivepressure is applied more copies of the plasmid integrate into the hostcell's chromosomal DNA. Apparently due to the similar structure-functionrelationships between the BK and SV40 T antigens and their respectivebinding sites, BK replication is also stimulated by SV40 T antigen. Toconstruct a derivative of plasmid pLPC that can exist as astably-maintained element in a transformed eukaryotic cell, the entireBK genome, as an EcoRI-linearized restriction fragment, was insertedinto the single EcoRI restriction site of plasmid pLPC. This insertionproduced two plasmids, designated pLPC4 and pLPC5, which differ onlywith respect to the orientation of the BK EcoRI fragment. A restrictionsite and function map of plasmid pLPC4 is presented in FIG. 11 of theaccompanying drawings, and the construction of plasmids pLPC4 and pLPC5is further described in Example 8.

Episomal maintenance of a recombinant DNA expression vector is notalways preferred over integration into the host cell chromosome.However, due to the absence of a selectable marker that functions ineukaryotic cells, the identification of stable, eukaryotic transformantsof plasmid pLPC is difficult, unless plasmid pLPC is cotransformed withanother plasmid that does comprise a selectable marker. Consequently,plasmid pLPC has been modified to produce derivative plasmids that areselectable in eukaryotic host cells.

This was done by ligating plasmid pLPC to a portion of plasmid pSV2hyg,a plasmid that comprises a hygromycin resistance-conferring gene. Arestriction site and function map of plasmid pSV2hyg, which can beobtained from the Northern Regional Research Laboratory (NRRL), Peoria,Ill. 61640, under the accession number NRRL B-18039, is presented inFIG. 12 of the accompanying drawings. Plasmid pSV2hyg was digested withrestriction enzyme BamHI, and the ˜2.5 kb BamHI restriction fragment,which comprises the entire hygromycin resistance-conferring gene, wasisolated, treated with Klenow enzyme (the large fragment produced uponsubtilisin cleavage of E. coli DNA polymerase I), and then ligated tothe Klenow-treated, ˜5.82 kb NdeI-StuI restriction fragment of plasmidpLPC to yield plasmids pLPChyg1 and pLPChyg2. Plasmids pLPChyg1 andpLPChyg2 differ only with respect to the orientation of the hygromycinresistance-conferring fragment. A restriction site and function map ofplasmid pLPChyg1 is presented in FIG. 13 of the accompanying drawings,and the construction protocol for plasmids pLPChyg1 and pLPChyg2 isdescribed in Example 9.

Plasmids pLPChyg1 and pLPChyg2 can be readily modified to contain the BKvirus genome. As stated above, expression of BK T-antigen in a host cellcontaining a plasmid comprising the BK T-antigen binding sites increasesthe copy number of the plasmid. If the plasmid also comprises aselectable marker, selection after T-antigen stimulated replication willresult in integration of more copies of the plasmid into the host'sgenomic DNA than would occur in the absence of T-antigen stimulatedreplication. Plasmids pLPChyg1 and pLPChyg2 each comprise two EcoRIsites, one in the HmR gene and the other in the pBR322-derived sequencesof the plasmid. Plasmid pLPChyg1 was partially digested with EcoRI toobtain cleavage only at the pBR322-derived EcoRI site and then ligatedwith EcoRI-digested BK virus DNA to yield plasmids pLPChT1 and pLPChT2,which differ only with respect to the orientation of the BK virus DNA.Plasmids pLPChT1 and pLPChT2 are useful derivatives of plasmid pLPChyg1(and analogous constructions can be made using plasmid pLPChyg2 asstarting material instead of pLPChyg1) for purposes of integrating highnumbers of copies of a protein C expression vector into the genome of aeukaryotic host cell.

Human protein C expression plasmids similar to plasmids pLPChyg1 andpLPChyg2 containing the dihydrofolate reductase (dhfr) gene wereconstructed by inserting the dhfr gene-containing. Klenow-treated ˜1.9kb BamHI restriction fragment of plasmid pBW32 into the ˜5.82 kbNdeI-StuI restriction fragment of plasmid pLPC. The resulting plasmids,designated as pLPCdhfr1 and pLPCdhfr2, differ only with respect to theorientation of the dhfr gene. The construction of these plasmids isdescribed in Example 11B.

Plasmid pLPChyg1 was further modified to introduce a dihydrofolatereductase (dhfr) gene. The dhfr gene is a selectable marker indhfr-negative cells and can be used to increase the copy number of a DNAsegment by exposing the host cell to increasing levels of methotrexate.The dhfr gene can be obtained from plasmid pBW32, a plasmid disclosedand claimed in U.S. patent application Ser. No. 769,298, filed Aug. 26,1985, and incorporated herein by reference. A restriction site andfunction map of plasmid pBW32 is presented in FIG. 14 of theaccompanying drawings. The construction protocol for plasmid pBW32 isdescribed in Example 10.

The dhfr gene-containing, ˜1.9 kb BamHI restriction fragment of plasmidpBW32 was isolated, treated with Klenow enzyme, and inserted intopartially-EcoRI-digested plasmid pLPChyg1 to yield plasmids pLPChd1 andpLPChd2. Plasmid pLPChyg1 contains two EcoRI restriction enzymerecognition sites, one in the hygromycin resistance-conferring gene andone in the plasmid pBR322-derived sequences. The fragment comprising thedhfr gene was inserted into the EcoRI site located in the pBR322-derivedsequences of plasmid pLPChyg1 to yield plasmids pLPChd1 and pLPChd2. Arestriction site and function map of plasmid pLPChd1 is presented inFIG. 15 of the accompanying drawings. The construction of plasmidspLPChd1 and pLPChd2, which differ only with respect to the orientationof the dhfr gene-containing DNA segment, is described in Example 11.

Plasmid pLPChd1 was modified to form plasmid phd, a plasmid thatcontains both the present BK enhancer-adenovirus late promoter cassetteand also the hygromycin resistance-conferring and dhfr genes. Toconstruct plasmid phd, plasmid pLPChd1 was prepared from dam E. colihost cells, digested with restriction enzyme BclI, and recircularized,thus deleting the human protein C-encoding DNA. Plasmid phd contains asingle BclI restriction enzyme recognition site, which is convenientlypositioned for the insertion of any sequence desired to be expressedfrom the BK enhancer-adenovirus late promoter of the present invention.A restriction site and function map of plasmid phd is presented in FIG.16 of the accompanying drawings, and the construction protocol forplasmid phd is described in Example 12.

Another expression vector that further exemplifies the present inventionand drives expression of human protein C is plasmid of pLPCE1A. PlasmidpLPCE1A contains the E1A gene of human adenovirus type 2, the geneproduct of which, as described above, increases the activity of the BKenhancer. Thus, transcription from a promoter in tandem with the BKenhancer increases in the presence of the E1A gene product. PlasmidpLPCE1A was constructed by ligating the E1A gene-containing, ˜1.8 kbBalI restriction fragment of human adenovirus-type-2 DNA with the ˜5.82kb NdeI-StuI restriction fragment of plasmid pLPC. A restriction siteand function map of plasmid pLPCE1A is presented in FIG. 17 of theaccompanying drawings, and the construction protocol for plasmid-pLPCE1Ais described in Example 13.

A variety of expression vectors of the present invention utilize the BKenhancer-adenovirus late promoter cassette to drive expression of tissueplasminogen activator (TPA) or modified TPA (MTPA). To construct suchvectors, plasmid pBW32 (FIG. 14) was digested with restriction enzymeBamHI, and the resultant ˜5.6 kb fragment was recircularized to yieldplasmid pBW32del. Plasmid pBW32del, which encodes modified TPA andcontains only one HindIII restriction site, was digested with HindIIIand then ligated with the ˜0.65 kb HindIII restriction fragment ofplasmid pBa18cat to yield plasmid pBLT. Plasmid pBa18cat comprises animproved BK enhancer-adenovirus late promoter cassette and is describedin Example 17. A restriction site and function map of plasmid pBLT ispresented in FIG. 18 of the accompanying drawings, and the consecutiveprotocol for plasmid pBLT is described in Example 14.

Selectable markers were introduced into BamHI-digested plasmid pBLT. Inone construction, the hygromycin resistance gene-containing, ˜2.5 kbBamHI restriction fragment of plasmid pSV2hyg was inserted to yieldplasmids pBLThyg1 and pBLThyg2, and in another construction, the dhfrgene-containing ˜1.9 kb BamHI restriction fragment of plasmid pBW32 wasinserted to yield plasmids, pBLTdhfr1 and pBLTdhfr2. The four plasmids,pBLThyg1, pBLThyg2, pBLTdhfr1, and pBLTdhfr2, differ only with respectto the type and/or orientation of the selectable marker. A restrictionsite and function map of each of plasmids pBLThyg1 and pBLTdhfr1 isrespectively presented in FIGS. 19 and 20 of the accompanying drawings.The construction protocol for plasmids pBLThyg1, pBLThyg2, pBLTdhfr1,and pBLTdhfr2 is described in Example 15.

Other expression vectors of the present invention that drive expressionof TPA or modified TPA were derived from plasmid pTPA103, anintermediate used in the construction of plasmid pBW32. The constructionprotocol for plasmid pTPA103 is described in Example 10, and arestriction site and function map of plasmid pTPA103 is presented inFIG. 14 of the accompanying drawings. To construct these derivatives, aBamHI restriction site was introduced immediately before the 5′ end ofthe TPA coding region of plasmid pTPA103. Plasmid pTPA103 was digestedwith restriction enzyme HgaI to isolate the ˜0.52 kb HgaI restrictionfragment that comprises the 5′ end of the TPA coding region. AfterKlenow treatment, the HgaI fragment was ligated to BamHI linkers,digested with restriction enzyme BamHI, and inserted into BamHI-digestedplasmid pBR322 to form plasmids pTPA601 and pTPA602. A restriction siteand function map of plasmid pTPA602, which differs from plasmid pTPA601only with respect to the orientation of the inserted BamHI restrictionfragment, is presented in FIG. 21 of the accompanying drawings.

Next, plasmid pTPA602 was digested with restriction enzymes BglII andSalI, and the resultant ˜4.2 kb BglII-SalI restriction fragment wasligated to the ˜2.05 kb SalI-BglII restriction fragment of plasmidpTPA103 to form plasmid pTPA603. Plasmid pTPA603 thus contains thecomplete coding sequence for TPA bounded by a BamHI restriction site onboth ends. A restriction site and function map of plasmid pTPA603 ispresented in FIG. 22 of the accompanying drawings. To construct aplasmid that is analogous to plasmid pTPA603 but that encodes a modifiedform of TPA, plasmid pTPA603 was digested with restriction enzymes BglIIand SstI, and the resultant ˜5.02 kb BglII-SstI fragment was ligated tothe ˜0.69 kb BglII-SstI restriction fragment of plasmid pBLT. Theresultant plasmid, designated as pMTPA603, was then digested withrestriction enzyme BamHI, and the resultant ˜1.35 kb fragment wasisolated. This fragment and the ˜1.90 kb BamHI restriction fragment ofplasmid pTPA603 were individually ligated in separate ligations toBclI-digested plasmid phd (FIG. 16) to form the respective plasmidsphdMTPA and phdTPA. Restriction site and function maps of plasmidsphdTPA and phdMTPA are respectively presented in FIGS. 23 and 24 of theaccompanying drawings. The construction of plasmids phdTPA and phdMTPA,beginning with the construction protocol for plasmid pTPA602, isdescribed in Example 16.

The present invention comprises a method for using the BK enhancer intandem with a eukaryotic promoter to drive transcription and expressionof DNA sequences in eukaryotic host cells that express animmediate-early gene of a large DNA virus. Skilled artisans willrecognize that virtually any eukaryotic promoter can be used in tandemwith the BK enhancer in the present method. For example, the SV40 earlyand late promoters, BK early and late promoters, early and latepromoters of any of the polyoma viruses or papovaviruses, herpes simplexvirus thymidine kinase promoter, interferon al promoter, mousemetallothionein promoter, promoters of the retroviruses, β-globinpromoter, promoters of the adenoviruses, sea urchin H2A promoter,conalbumin promoter, ovalbumin promoter, mouse β-globin promoter, humanβ globin promoter, and the Rous sarcoma virus long terminal repeatpromoter, can all serve as the eukaryotic promoter in the method of thepresent invention. Moreover, any sequence containing a transcriptionstart site, composed of a “TATA”-like sequence with or without anupstream “CAAT” sequence, can serve as the promoter in the presentinvention. Such promoters can be utilized in the present method byconventionally inserting the promoters into expression vectorscomprising the BK enhancer as exemplified herein using the adenovirus-2late promoter, which is the preferred eukaryotic promoter for use in thepresent method.

The BK enhancer used in the vectors herein that exemplify the presentinvention was isolated from the prototype strain of BK virus (ATCCVR-837). However, a number of BK virus variants have been isolated anddescribed. Gardner et al., 1971, The Lancet 1:1253, (see also Gardner,1973, Brit. Med. J. 1:77-78) described the first isolation of a BKvirus, and the Gardner strain is thus referred to as the prototype orwild-type BK virus. The Gardner strain of BK virus (FIG. 1) is availablefrom the ATCC under the accession number ATCC VR-837. In fact, when ATCCVR-837 was obtained for use in constructing the vectors of theinvention, it was observed that BK variants were present in thepopulation of viruses. Others have observed this phenomenon, i.e., Chukeet al., 1986, J. Virology 60(3):960. Neither the method of using the BKenhancer in tandem with a eukaryotic promoter to drive expression ofuseful substances, such as nucleic acid and protein, in the presence ofan immediate-early gene product of a large DNA virus nor any othermethod of the present invention is limited to the Gardner strain or aparticular BK variant, although the enhancer of the prototype strain ispreferred. The following Table lists a representative number of BKvariants that can be used in the methods of the present invention. Inaddition, a BK-like virus (simian agent 12) contains enhancer elementshomologous to the BK enhancer and can be used in the methods of thepresent invention. The enhancer elements of simian agent 12 aredescribed in Cunningham et al., 1985, J. Virol. 54:483-492 and, forpurposes of the present invention, are BK enhancer variants.

TABLE 1 BK Variants Description Strain designation (relative towild-type) Reference BKV(DUN) BKV(DUN) contains an ˜40 bp dele- ViralOncology, 1980 tion at 0.7 m.u, just to the late (Raven Press, N.Y.; ed.coding side of the viral enhancer G. Klein), pp. 489-540. core. BK(GS)and BK(MM) These variants have numerous base Pater et al., 1979, J.differences that include rearrange- Viral. 32: 220-225; ments andduplications in the con- Seif et al., 1979, Cell trol region; somedifferences occur 18:963-977; in the enhancer Yang et al., 1979 Nuc.Acids Res. 7:651-668; and Pater et al., 1979, Virology 131:426-436.BK(II) Minor differences in restriction Pauw et al., 1978, endonucleasepatterns. Arch. Viral. 57:35-42. BK(GS) and BK(MG) These variants arecomposed of Pater et al., 1980, J. two complementary defective mole-Virol. 36:480-487; cules, both of which are required Pater et al., 1981,J. for infectivity and differ Virol. 39:968-972; extensively innucleotide Pater et al., 1983, sequence from prototype BK virus. Virol.131:426-436. pm522 Spontaneous mutation during Watanabe et al., 1982,propagation led to differences J. Virol. 42:978-985; in host range andtransforming Watanabe et al., 1984, potential, perhapes due to a J.Virol. 51:1-6. deletion of two of the three enhancer repeats and thepresence of two sets of shorter 37 bp repeats. tr530 Spontaneousmutation during Watanabe et al., 1984, tr531 propagation of recombinantJ. Virol. 51:1-6. tr532 BK virus containing the pm522 enhancer regionand having further duplications of short segments originating from thepm522 sequence. BKV9 Viable variant of BK virus iso- Chuke et al., 1986.lated from a preparation of J. Virol. 60:960-971. prototype (wt) BKvirus contains an incomplete enhancer repeat and duplication ofsequences to the late side of the enhancer. BK virus-IR BK virus variantisolated from a Pagnani et al., 1986, human tumor containing insertionsJ. Virol. 59:500-505. and rearrangements in the enhancer region. Thisvirus has an altered transformation phenotype.

Skilled artisans will understand that a variety of eukaryotic host cellscan be used in the present method, so long as the host cell expresses animmediate-early gene product of a large DNA virus. Because theimmediate-early gene product can be introduced into host cells by manymeans, such as transformation with a plasmid or other vector, virtuallyany eukaryotic cell can be used in the present method. Human cells arepreferred host cells in the method of the present invention, becausehuman cells are the natural host for BK virus and may contain cellularfactors that serve to stimulate the BK enhancer. While human kidneycells are especially preferred as host cells, the adenovirus5-transformed human embryonic kidney cell line 293, which expresses theE1A gene product, is most preferred and is available from the ATCC underthe accession number ATCC CRL 15753.

The 293 cell line is preferred not only because 293 cells express theE1A gene product but also because of the ability of the 293 cells toγ-carboxylate and otherwise properly process complex gene products suchas protein C. “γ-Carboxylation” refers to a reaction in which a carboxylgroup is added to a glutamic acid residue at the γ-carbon, and aγ-carboxylated protein is a protein in which some amino acid residueshave undergone γ-carboxylation. Kidney cells normally γ-carboxylate andotherwise process certain proteins, but 293 cells are transformed withadenovirus, which generally results in a loss of specialized functions.Consequently, the present invention also comprises an improvement in themethod for producing a protein that is naturally gamma carboxylated,properly folded, and processed wherein said protein is encoded in arecombinant DNA vector such that said protein is expressed when aeukaryotic host cell containing said vector is cultured under suitableexpression conditions, wherein the improvement comprises: (a) insertingsaid vector into an adenovirus-transformed, human embryonic kidney cell;and (b) culturing said host cell of step a) under growth conditions andin media containing sufficient vitamin K for carboxylation. The 293N3Sderivative of the 293 cell line is also suitable for use in the presentinvention and is able to grow in suspension culture as described inGraham, 1987, J. Gen. Virol. 68:937.

This method of producing a γ-carboxylated protein is not limited toadenovirus-transformed human embryonic kidney cells. Instead, the methodof producing a γ-carboxylated protein is broadly applicable to alladenovirus-transformed host cells. Those skilled in the art alsorecognize that the method can be practiced by first transforming aeukaryotic cell with an expression vector for a γ-carboxylated proteinand then transforming the resulting transformant with adenovirus. HaroldGinsberg, in The Adenoviruses (1984, Plenum Press, New York), describesa number of adenoviruses and methods of obtaining adenovirus-transformedhost cells. One especially preferred adenovirus-transformed host cellfor purposes of expressing a γ-carboxylated protein encoded on arecombinant DNA expression vector is the Syrian hamster cell lineAV12-664 (hereinafter AV12). The AV12 cell line was constructed byinjecting adenovirus type 12 into the scruff of the neck of a Syrianhamster and isolating cells from the resulting tumor. The AV12 cell lineis a preferred host for purposes of producing a γ-carboxylated protein.Examples of γ-carboxylated proteins include, but are not limited to,Factor VII, Factor IX, Factor V, protein C, protein S, protein Z, andprothrombin. Example 19, below, illustrates the advantages of using anadenovirus-transformed host cell for expression of recombinantγ-carboxylated proteins.

In addition to the increased efficiency of γ-carboxylation of proteins,the present invention further provides methods for the production ofmolecules never before encountered in nature. The gene encoding humanprotein C is disclosed and claimed in Bang et al., U.S. Pat. No.4,775,624, issued Oct. 4, 1988, the entire teaching of which is hereinincorporated by reference. Human protein C is a glycoprotein whichcontains four potential sites for the addition of N-linkedoligosaccharides. These glycosylation sites occur at the asparagineresidues found at positions 97, 248, 313 and 329 of the human protein Cmolecule. The carbohydrate residues attached to human protein Cspecifically affect the functional activities (both anticoagulent andamidolytic) of the molecule. Human protein C which is totallydeglycosylated has no functional activity. The functional activity ofrecombinant human protein C from adeno-transformed Baby Hamster Kidney(BHK) cells is about 5-10% lower than fully glycosylated human protein Cderived from plasma. However, recombinant human protein C from 293 cellshas a functional activity which is 30-40% greater than plasma-derivedhuman protein C.

The differences in functional activities between plasma HPC, rHPC fromBHK cells and rHPC from 293 cells are not due to any significantdifferences in the γ-carboxyglutamate or β-hydroxyaspartate content ofthe molecules. While all three of the molecules appear to be fullyγ-carboxylated, the rHPC from 293 cells demonstrates much higherfunctional activity. The reason for the different activities lies in theglycosyl content of the separate molecules as summarized in thefollowing table.

TABLE 2 moles sugar/mole of HPC rHPC- rHPC- Sugar Plasma HPC 293 cellsBHK cells Fucose (Fuc) 0.9 4.8 4.0 N-acetylgalactosamine (GalNAc) 0 2.60.62 N-acetylglucosamine (GlcNAc) 13.8 12.4 16.8 Galactose (Gal) 9.3 6.010.6 Mannose (Man) 9.1 8.5 10.2 N-acetylneuraminic acid 10.2 5.4 10.9(NeuAc)(Sialic acid)

This glycosyl content data predicts that for plasma HPC and BHK-derivedrHPC the oligosaccharides are predominantly of N-linked complextriantennary structure as shown below:

The glycosyl content for rHPC produced in 293 cells, however, predictsthat most oligosaccharide chains are predominantly of the N-linkedcomplex biantennary structure as shown below:

The N-acetylgalactose residues present in rHPC derived from 293 cellsare totally in N-linked oligosaccharide structures and not o-linkedbecause they can be totally released by N-glycanose digestion. The totalremoval of sialic acid from HPC with neuraminidase resulted in a 50%increase in amidolytic activity and a 250-300% increase in anticoagulentactivity, therefore, as the sialic acid content of the molecule islowered, the functional activity of the molecule is increased.

However, the removal of sialic acid and the concomitant exposure of thegalactose residue on the non-reducing end of oligosaccharides ofglycoproteins results in general, in a tremendous increase in theclearance rate of the glycoprotein by the liver, therefore asialylatedglycoproteins are not pharmaceutically preferred. In rHPC derived from293 cells, the lowering of the sialic acid content is matched with aproportional lowering of the galactosyl content. The ratio ofgalactose:sialic acid is the same in plasma HPC, rHPC-BHK and rHPC-293and is close to 1:1 in all three molecules. The data demonstrates thatthere are few or no galactosyl residues at the non-reducing end of theoligosaccharides in the rHPC from 293 cells. This lower sialic acidcontent in rHPC from 293 cells is consistent with the interpretation ofless branching in the N-linked oligosaccharides. This novel structureresults in a molecule with increased activity which should not have anincreased rate of clearance from the blood. As the biosynthesis ofoligosaccharides on glycoproteins is in part regulated by the“machinary” of the cells from which the glycoproteins are secreted, themethods of the present invention allow for the production of novelglycoprotein molecules from a wide variety of host cells. In particular,recombinant human protein C produced in AV12 cells also displays novelglycosylation patterns.

The novel BK enhancer-eukaryotic promoter constructions described inExample 17 were constructed using a method for improving the activity ofthe BK enhancer with respect to a eukaryotic promoter. Such methodcomprises placing the BK enhancer within 0 to 300 nucleotides upstreamof the 5′ end of the CAAT region or CAAT region equivalent of theeukaryotic promoter used in tandem with the BK enhancer. The improvedcassettes produced by this method comprise an important embodiment ofthe present invention. Use of the improved cassettes is not limited tohost cells that express E1A or a similar gene product, although thepreferred use does involve stimulation of the improved cassette by animmediate-early gene product of a large DNA virus.

Other viral gene products, such as the VA gene product of adenovirus,can be used to increase the overall efficiency of the present method forusing the BK enhancer to promote transcription and expression ofrecombinant genes in eukaryotic host cells. The VA gene productincreases the translation efficiency of mRNA molecules that contain thetripartite leader of adenovirus (Kaufman, 1985, PNAS, 82:689-693, andSvensson and Akusjarul, 1985, EMBO, 4:957-964). The vectors of thepresent invention can be readily modified to encode the entiretripartite leader of adenovirus; however, as demonstrated in Example 18,the present invention encompasses the use of the VA gene product toincrease translation of a given mRNA that only contains the first partof the adenovirus tripartite leader.

The sequence of the tripartite leader of adenovirus is depicted below:

   *---------------  First Part  --------------**-----------------Second5′-ACUCUCUUCCGCAUCGCUGUCUGCGAGGGCCAGCUGUUGGGCUCGCGGUUGAGGACAACUCUUCPart -------------------------------------------** ------------- ThirdGCGGUCUUUCCAGUACUCUUGGAUCGGAAACCCGUCGGCCUCCGAACGUACUCCGCCACCGAGGGACC                                                                   *Part-----------------------------------------------------------------UGAGCGAGUCCGCAUCGACCGGAUCGGAAAACCUCUCGAGAAAGGCGUCUAACCAGUCACAGUCGCA-3′,

wherein A is riboadenyl, G is riboguanyl, C is ribocytidyl, and U isuridyl. As encoded in adenovirus DNA, the tripartite leader isinterrupted by large introns. The presence of these introns or portionsof the introns does not adversely affect expression levels. Plasmidsp4-14 and p2-5 of the present invention contain the tripartite leader ofadenovirus and are described more fully in Example 20, below.

Many of the illustrative vectors of the invention, such as plasmidspBLcat and pLPC, contain only the first part of the tripartite leader ofadenovirus. As used herein, the “first part” of the tripartite leader ofadenovirus, when transcribed into mRNA, comprises at least the sequence:

5′-ACUCUCUUCCGCAUCGCUGUCUGCGAGGGCCAG-3′.

Thus, the present invention comprises an improvement in the method forproducing a useful substance in a eukaryotic host cell that istransformed with a recombinant DNA vector that contains both aeukaryotic promoter and a DNA sequence that encodes said usefulsubstance, said sequence being positioned for expression from saidpromoter, and wherein said cell containing said vector is cultured underconditions suitable for expression of said useful substance, wherein theimprovement comprises:

(a) incorporating DNA that encodes the first part of the tripartiteleader of an adenovirus into said vector such that, upon transcription,the mRNA produced encodes said useful product and, at the 5′ end,contains said first part of the tripartite leader;

(b) providing said cell containing the vector of step a) with a DNAsequence that codes for the expression of a VA gene product of saidadenovirus; and

(c) culturing said cell of step b) under conditions suitable forexpressing said VA gene product and for stimulating translation of saidmRNA,

subject to the limitation that said MRNA does not contain the entiretripartite leader of said adenovirus.

Plasmids coding for VA have been constructed from adenovirus DNA. Arestriction fragment of ˜1723 bp, defined by a SalI site (at nucleotide9833) and a HindIII site (at nucleotide 11556), was isolated fromadenovirus-2 DNA and cloned into HindIII-SalI-digested plasmid pBR322,thus replacing the ˜622 bp SalI-HindIII fragment of pBR322, to constructplasmid pVA. A plasmid coding for neomycin resistance and VA has beenconstructed by isolating a ˜1826 bp NruI fragment from plasmid pVA andinserting that fragment into Klenow-treated, BamHI-digested plasmidpSVNeo (available from BRL). The resultant plasmid, designated pVA-Neo,can be used to insert the VA gene into any cell line by selection ofneomycin (G418) resistance after transformation.

The VA gene product of adenovirus, however, may exert its greatestpositive effect on expression of recombinant genes containing either thefirst part of the tripartite leader of adenovirus, or the entiretripartite leader, in the first few days following transformation of thehost cell with a VA-encoding vector. Subsequent expression of the VAgene product in the host cell after the first few days may not giveoptimal expression levels. However, presence of the first part of thetripartite leader on the expression vector and resulting message willlead to increased expression of the product encoded by the mRNA, even inthe absence of the VA gene product, in comparison to expression vectorsand mRNA molecules that lack the first part of the tripartite leader.

The T antigen of SV40, BK virus, or any other polyoma-virus can also beused with the vectors of the present invention to increase promoteractivity and/or increase copy number of the plasmid by stimulatingreplication. SV40 T antigen stimulates transcription from both theadenovirus and BK late promoters. By including T-antigen-codingsequences on the expression vectors of the present invention or bycotransfection of the vectors with a plasmid(s) carryingT-antigen-coding sequences, amplification of copy number can be obtainedprior to the application of selective pressure as outlined in Example18. This will allow for high copy number integration of the expressionvector.

Thus, in the preferred embodiment of the present invention, therecombinant DNA expression vector comprises the BK enhancer of theprototype strain positioned less than 300 nucleotides upstream of theadenovirus late promoter, which itself is positioned to drive expressionof a gene that encodes at least the first part of the tripartite leaderand a useful substance. This preferred vector is used to transform humanembryonic kidney 293 cells that have been modified, either before orafter transformation with the expression vector, to express the VA geneproduct of an adenovirus. For stable transformants, however, presence ofthe VA gene product may not be desired.

The present invention also concerns a method of amplifying genes inprimate cells. DNA encoding a directly selectable marker, the murinedihydrofolate reductase gene and a structural polypeptide is introducedinto primate cells. Those cells which contain the directly selectablemarker are then reisolated and treated with progressively increasingamounts of methotrexate to amplify the genes for dihydrofolate reductaseand the structural polypeptide. This method allows for a significantincrease in the amount of the structural polypeptide gene that can be inthe cells.

Many gene products require extensive post-translated modification forfunctional activity. As some cell lines do not efficiently modify suchgene products, it is advantageous to express these genes in those celllines which can perform such modifications. Human protein C is one geneproduct which requires both gamma carboxylation and the removal of apropiece after the translation of the gene. These post-translationalmodifications occur most efficiently in primate cells, yet the genesencoding such gene products cannot be directly amplified in primatecells.

The most common system for gene amplification employs the murinedihydrofolate reductase (dhfr) gene in dhfr deficient cell lines.Dihydrofolate reductase reduces folic acid to tetrahydrofolic acid,which is involved in the synthesis of thymidylic acid. Methotrexatebinds to dihydrofolate reductase, thereby preventing the biosynthesis ofthymidylic acid. Dihydrofolate reductase deficient cells, therefore,cannot survive in an environment which does not contain thymidylic acid,while the presence of methotrexate in the culture media requires aconcomitant increase in the amount of non-bound dihydrofolate reductasefor cell survival.

Primate cells, on the other hand, which are most efficient in thepost-translational modification of certain polypeptides, also contain aconstitutive dhfr gene. The presence of the constitutive dhfr geneprevents the direct selection of transformants and amplifications ofgenes using methotrexate. The method of the present invention allows forthe direct selection of transformants using a separate selectablemarker, such as the hygromycin resistance-conferring gene or theneomycin resistance-conferring gene. Following this direct selection,the genes may then be amplified by progressively increasing the level ofmethotrexate in the culture media. Many cells which demonstrate anincreased level of dhfr gene copy number as well as any increase in thecopy number of the structural polypeptide gene.

The method of gene amplification in primate cells is in no way dependentupon any given means for the introduction of the DNA into the cells.Those skilled in the art recognize that DNA may be introduced into cellsby electroporation, microinjection, transformation or transfection.Furthermore, the DNA can either be linear or circular. The gene encodinga selectable marker does not need to be an antibiotic resistanceconferring gene. Skilled artisans understand that any means for directselection may be utilized in the present invention. For example, a geneencoding an antigenic determinant could be introduced into a cell line,and cells containing this determinant could be easily selected usingimmunological methods which are well known in the art.

The directly selectable marker gene, the dhfr gene and the structuralpolypeptide gene do not need to be introduced into the cell on the samepiece of DNA. For example, the directly selectable marker may betransfected into the cell on one plasmid, while the dhfr and structuralpolypeptide genes may be transfected into the cell on a separateplasmid. This occurs when the hygromycin resistance conferring gene istransfected into 293 cells via plasmid pLPGhyg, while the dhfr and humanprotein C genes are transfected into the same cells via plasmidpLPCdhfr. Alternatively, the dhfr and human protein C genes can beintroduced into plasmid pLPChyg-transfected 293 cells via plasmid p4-14.The neomycin-resistance conferring gene can be used in place of thehygromycin resistance-conferring gene, in which case plasmid pSV2neo isintroduced into the cell line rather than plasmid pLPChd. In addition toco-transfection with different plasmids, the directly selectable markergene, the dhfr gene and the structural polypeptide gene can all beintroduced into the host cell on one plasmid. This is exemplified by thetransfection of cell line 293 with plasmid pLPChd. Furthermore, othertypes of primate cells, such as the monkey kidney MK2 cell line (ATCCCCL7), may be used in the method of the present invention.

The following Examples more fully describe the methods, compounds, andrecombinant organisms of the present invention. Those skilled in the artwill recognize that the particular reagents, equipment, and proceduresdescribed in the Examples are merely illustrative and do not limit thepresent invention.

EXAMPLE 1 Preparation of BK Virus DNA

BK virus is obtained from the American Type Culture Collection under theaccession number ATCC VR-837. The virus is delivered in freeze-driedform and resuspended in Hank's balanced salts (Gibco, 3175 Staley Road,Grand Island, N.Y. 14072) to a titer of about 10⁵ plaque-forming units(pfu)/ml. The host of choice for the preparation of BK virus DNA isprimary human embryonic kidney (PHEK) cells, which can be obtained fromFlow Laboratories, Inc., 7655 Old Springhouse Road, McLean, Va. 22101,under catalogue number 0-100 or from M. A. Bioproducts under cataloguenumber 70-151.

About five 75 mm² polystyrene flasks comprising confluent monolayers ofabout 10⁶ PHEK cells are used to prepare the virus. About 1 ml of BKvirus at a titer of 10⁵ pfu/ml is added to each flask, which is thenincubated at 37° C. for one hour, and then, fresh culture medium(Dulbeccols Modified Eagle's Medium, Gibco, supplemented with 10% fetalbovine serum) is added, and the infected cells are incubated at 37° C.for 10-14 days or until the full cytopathogenic effect of the virus isnoted. This cytopathogenic effect varies from cell line to cell line andfrom virus to virus but usually consists of cells rounding up, clumping,and sloughing off the culture disk.

The virus is released from the cells by three freeze-thaw cycles, andthe cellular debris is removed by centrifugation at 5000×g. The virus is1 liter of supernatant fluid is precipitated and collected by theaddition of 100 g of PEG-6000, incubation of the solution for 24 hoursat 4° C., and centrifugation at 5000×g for 20 minutes. The pellet isdissolved in 0.1×SSC buffer (1×SSC=0.15M NaCl and 0.015M NaCitrate,pH=7) at 1/100th of the original volume. The virus suspension is layeredonto a 15 ml solution of saturated KBr in a tube, which is centrifugedat 75,000×g for 3 hours. Two bands are evident in the KBr solution aftercentrifugation. The lower band, which contains the complete virion, iscollected and desalted on a Sephadex® G-50 column (Sigma Chemical Co.,P.O. Box 14508, St. Louis, Mo. 63178) using TE (10 mM Tris-HCl, pH=7.8,and 1 mM EDTA) as an elution buffer.

Sodium dodecyl sulfate (SDS) is added to the solution of purifiedvirions obtained from the column to a concentration of 1%; pronase isadded to a concentration of 100 μg/ml, and the solution is incubated at37° C. for 2 hours. Cesium chloride is then added to the solution to adensity of 1.56 g/ml, and ethidium bromide is added to the solution to afinal concentration of 100 μg/ml. The solution is centrifuged in aSorvall (DuPont Inst. Products, Biomedical Division, Newton, Conn.06470) 865 rotor or similar vertical rotor at 260,000×g for 24 hours.After centrifugation, the band of virus DNA is isolated and extractedfive times with isoamyl alcohol saturated with 100 mM Tris-HCl, pH=7.8.The solution of BK virus DNA is then dialyzed against TE buffer untilthe 260 nm/280 nm absorbance ratio of the DNA is between 1.75 and 1.90.The DNA is precipitated by adjusting the NaCl concentration to 0.15M,adding two volumes of ethanol, incubating the solution at −70° C. for atleast 2 hours, and centrifuging the solution at 12,000×g for 10 minutes.The resulting pellet of BK virus DNA is suspended in TE buffer at aconcentration of 1 mg/ml.

EXAMPLE 2 Construction of Plasmids pBKE1 and pBKE2

About one μg of the BK virus DNA prepared in Example 1 in one μl of TEbuffer was dissolved in 2 μl of 10× EcoRI buffer (1.0M Tris-HCl, pH=7.5;0.5M NaCl; 50 mM MgCl₂; and 1 mg/ml BSA) and 15 μl of H₂O. About 2 μl(˜10 units; all enzyme units referred to herein, unless otherwiseindicated, refer to the unit definitions of New England Biolabs, 32Tozer Road, Beverly, Mass. 01915-9990, although the actual source of theenzymes may have been different) of restriction enzyme EcoRI were addedto the solution of DNA, and the resulting reaction was incubated at 37°C. for two hours.

About 1 μg of plasmid pUC8 (available from Pharmacia P-L Biochemicals,800 Centennial Ave., Piscataway, N.J. 08854) in 1 μl of TE buffer wasdigested with EcoRI in substantial accordance with the procedure used toprepare the EcoRI-digested BK virus DNA. The EcoRI-digested plasmid pUC8DNA was diluted to 100 μl in TE buffer; ˜0.06 units of calf-intestinalalkaline phosphatase were added to the solution, and the resultingreaction was incubated at 37° C. for 30 minutes. The solution wasadjusted to contain 1×SET (5 mM Tris-HCl, pH=7.8; 5 mM EDTA; and 150 mMNaCl), 0.3M NaOAc, and 0.5% SDS and then incubated at 65° C. for 45minutes. The phosphatase treatment prevents the pUC8 DNA from selfligating.

The EcoRI-digested BK virus and plasmid pUC8 DNA were extracted firstwith buffered phenol and then with chloroform. The DNA was collected byadjusting the NaCl concentration of each DNA solution to 0.25M, addingtwo volumes of ethanol, incubating the resulting mixtures in a dryice-ethanol bath for 5 minutes, and centrifuging to pellet the DNA. Thesupernatants were discarded, and the DNA pellets were rinsed with 70%ethanol, dried, and resuspended in 10 μl and 30 μl of TE buffer for theBK and plasmid pUC8 samples, respectively.

About 3 μl of H₂O and 1 μl of 10× ligase buffer (0.5M Tris-HCl, pH=7.8;100 mM MgCl₂; 200 mM DTT; 10 mM ATP; and 0.5 mg/ml BSA) were added to amixture of 2 μl of the EcoRI-digested BK virus and 1 μl of theEcoRI-digested plasmid pUC8 DNA. One μl (˜1000 units) of T4 DNA ligasewere added to the solution of DNA, and the resulting reaction wasincubated at 16° C. overnight. The ligated DNA constituted the desiredplasmids pBKE1 and pBKE2, which differ only with respect to theorientation of the inserted BK virus DNA. A restriction site andfunction map of plasmid pBKE1 is presented in FIG. 2 of the accompanyingdrawings.

A 50 ml culture of E. coli K12 JM103, available from Pharmacia P-LBiochemicals, in L-broth was grown to an optical density at 650nanometers (O.D.₆₅₀) of approximately 0.4 absorbance units. The culturewas chilled on ice for ten minutes, and the cells were collected bycentrifugation. The cell pellet was resuspended in 25 ml of cold 100 mMMgCl₂ and incubated on ice for 25 minutes. The cells were once againpelleted by centrifugation, and the pellet was resuspended in 2.5 ml ofcold 100 mM CaCl₂ and incubated for 30 minutes on ice. After theincubation, the cells are competent for the uptake of transforming DNA.

Two hundred μl of this cell suspension were mixed with the ligated DNAprepared above and incubated on ice for 30 minutes. At the end of thisperiod, the cells were placed in a water bath at 42° C. for 2 minutesand then returned to the ice for an additional 10 minutes. The cellswere collected by centrifugation and resuspended in one ml of L brothand incubated at 37° C. for 1 hour.

Aliquots of the cell mixture were plated on L-agar (L broth with 15grams of agar per liter) plates containing 100 μg ampicillin/ml, 40 μgX-gal/ml, and 40 μg IPTG/ml. The plates were incubated at 37° C.overnight. Colonies that contain a plasmid without an insert, such as E.coli K12 JM103/pUC8, appear blue on these plates. Colonies that containa plasmid with an insert, such as E. coli K12 JM103/pBKE1, are white.Several white colonies were selected and screened by restriction enzymeanalysis of their plasmid DNA for the presence of the ˜5.2 kb EcoRIrestriction fragment of BK virus. Plasmid DNA was obtained from the E.coli K12 JM103/pBKE1 and E. coli K12 JM103/pBKE2 cells in substantialaccordance with the procedure for isolating plasmid DNA that isdescribed in the following Example, although the procedure is done on asmaller scale, and the CsCl gradient steps are omitted, when the plasmidDNA is isolated only for restriction enzyme analysis.

EXAMPLE 3 Construction of Plasmids pBKneo1 and pBKneo2

E. coli K12 HB101/pdBPV-DIMTneo cells are obtained in lyophil form fromthe American Type Culture Collection under the accession number ATCC37224. The lyophilized cells are plated on L-agar plates containing 100μg/ml ampicillin and incubated at 37° C. to obtain single colonyisolates.

One liter of L broth (10 g tryptone, 10 g NaCl, and 5 g yeast extractper liter) containing 50 μg/ml ampicillin was inoculated with a colonyof E. coli K12 HB101/pdBPV-MMTneo and incubated in an air-shaker at 37°C. until the O.D.₅₉₀ was ˜1 absorbance unit, at which time 150 mg ofchloramphenicol were added to the culture. The incubation was continuedfor about 16 hours; the chloramphenicol addition inhibits proteinsynthesis, and thus inhibits further cell division, but allows plasmidreplication to continue.

The culture was centrifuged in a Sorvall GSA rotor (DuPont Co.,Instrument Products, Biomedical Division, Newtown, Conn. 06470) at 6000rpm for 5 minutes at 4° C. The resulting supernatant was discarded, andthe cell pellet was washed in 40 ml of TES buffer (10 mM Tris-HCl,pH=7.5; 10 mM NaCl; and 1 mM EDTA) and then repelleted. The supernatantwas discarded, and the cell pellet was frozen in a dry ice-ethanol bathand then thawed. The thawed cell pellet was resuspended in 10 ml of asolution of 25% sucrose and 50 mM EDTA. About 1 ml of a 5 mg/ml lysozymesolution; 3 ml of 0.25M EDTA, pH=8.0; and 100 μl of 10 mg/ml RNAse Awere added to the solution, which was then incubated on ice for 15minutes. Three ml of lysing solution (prepared by mixing 3 ml 10%Triton-X 100; 75 ml 0.25M EDTA, pH=8.0; 15 ml of 1M Tris-HCl, pH=8.0;and 7 ml of water) were added to the lysozyme-treated cells, mixed, andthe resulting solution incubated on ice for another 15 minutes. Thelysed cells were frozen in a dry ice-ethanol bath and then thawed.

The cellular debris was removed from the solution by centrifugation at25,000 rpm for 40 minutes in an SW27 rotor (Beckman, 7360 N. LincolnAve., Lincolnwood, Ill. 60646) and by extraction with buffered phenol.About 30.44 g of CsCl and ˜1 ml of a 5 mg/ml ethidium bromide solutionwere added to the cell extract, and then, the volume of the solution wasadjusted to 40 ml with TES buffer. The solution was decanted into aVTi50 ultra-centrifuge tube (Beckman), which was then sealed andcentrifuged in a VTi50 rotor at 42,000 rpm for ˜16 hours. The resultingplasmid band, visualized with ultraviolet light, was isolated and thenplaced in a Ti75 tube and rotor (Beckman) and centrifuged at 50,000 rpmfor 16 hours. Any necessary volume adjustments were made using TEScontaining 0.761 g/ml CsCl. The plasmid band was again isolated,extracted with salt-saturated isopropanol to remove the ethidiumbromide, and diluted 1:3 with TES buffer. Two volumes of ethanol werethen added to the solution, which was then incubated overnight at −20°C. The plasmid DNA was pelleted by centrifuging the solution is an SS34rotor (Sorvall) for 15 minutes at 10,000 rpm.

The ˜1 mg of plasmid pdBPV-MMTneo DNA obtained by this procedure wassuspended in 1 ml of TE buffer and stored at −20° C. The foregoingplasmid isolation procedure is generally used when large amounts of verypure plasmid DNA are desired. The procedure can be modified to rapidlyobtain a smaller, less pure amount of DNA, such as is needed whenscreening transformants for the presence of a given plasmid, by usingonly about 5 ml of cultured cells, lysing the cells in an appropriatelyscaled-down amount of lysis buffer, and replacing the centrifugationsteps with phenol and chloroform extractions.

About 5 μg (5 μl) of the plasmid pdBPV-MMTneo DNA prepared above andfive μg (5 μl) of the BK virus DNA prepared in Example 1 were eachdigested at 37° C. for 2 hours in a solution containing 2 μl of 10×BamHI buffer (1.5M NaCl; 60 mM Tris-HCl, pH=7.9; 60 MM MgCl₂; and 1mg/ml BSA), 1 μl of restriction enzyme BamHI, and 7 μl of H₂O. Thereaction was stopped by an extraction with an equal volume of phenol,followed by two extractions with chloroform. Each BamHI-digested DNA wasthen precipitated, collected by centrifugation, and resuspended in 5 μlof H₂O.

About 1 μl of 10× ligase buffer was added to a mixture of BamHI-digestedplasmid pdBPV-MMTneo (1 μl) and BamHI-digested BK virus DNA (1 μl).After 1 μl (˜1000 units) of T4 DNA ligase and 6 μl of H₂O were added tothe mixture of DNA, the resulting reaction was incubated at 16° C.overnight. The ligated DNA constituted the desired plasmids pBKneo1 andpBKneo2, which differ only with respect to the orientation of the BKvirus DNA. A restriction site and function map of plasmid pBKneo1 ispresented in FIG. 3 of the accompanying drawings.

E. coli K12 HB101 cells are available in lyophilized form from theNorthern Regional Research Laboratory under the accession number NRRLB-15626. E. coli K12 HB101 cells were cultured, made competent fortransformation, and transformed with the ligated DNA prepared above insubstantial accordance with the procedure of Example 2. The transformedcells were plated on L-agar plates containing 100 μg/ml ampicillin. E.coli K12 HB101/pBKneo1 and E. coli K12/pBKneo2 transformants wereidentified by their ampicillin-resistant phenotype and by restrictionenzyme analysis of their plasmid DNA.

EXAMPLE 4 Construction of Plasmid pBLcat

A. Construction of Intermediate Plasmid pLPcat

The virion DNA of adenovirus 2 (Ad2) is a double-stranded linearmolecule about 35.94 kb in size. The Ad2 late promoter can be isolatedon an ˜0.316 kb AccI-PvuII restriction fragment of the Ad2 genome; this˜0.32 kb restriction fragment corresponds to the sequence betweennucleotide positions 5755 and 6071 of the Ad2 genome. To isolate thedesired ˜0.32 kb AccI-PvuII restriction fragment, Ad2 DNA is firstdigested with restriction enzyme BalI, and the ˜2.4 kb BalI restrictionfragment that comprises the entire sequence of the ˜0.32 kb AccI-PvuIIrestriction fragment is isolated. Then, the ˜2.4 kb BalI restrictionfragment is digested with AccI and PvuII to obtain the desired fragment.

About 50 μg of Ad2 DNA (available from BRL) are dissolved in 80 μl ofH₂O and 10 μl of 10× BalI buffer (100 mM Tris-HCl, pH=7.6; 120 mM MgCl₂;100 mM DTT; and 1 mg/ml BSA). About 10 μl (˜20 units) of restrictionenzyme BalI are added to the solution of Ad2 DNA, and the resultingreaction is incubated at 37° C. for 4 hours.

The BalI-digested DNA is loaded onto an agarose gel and electrophoreseduntil the restriction fragments are well separated. Visualization of theelectrophoresed DNA is accomplished by staining the gel in a dilutesolution (0.5 μg/ml) of ethidium bromide and exposing the stained gel tolong-wave ultraviolet (UV) light. One method to isolate DNA from agaroseis as follows. A small slit is made in the gel in front of the desiredfragment, and a small piece of NA-45 DEAE membrane (Schleicher andSchuell, Keene, N.H. 03431) is placed in each slit. Upon furtherelectrophoresis, the DNA non-covalently binds to the DEAE membrane.After the desired fragment is bound to the DEAE membrane, the membraneis removed and rinsed with low-salt buffer (100 mM KCl; 0.1 mM EDTA; and20 mM Tris-HCl, pH=8). Next, the membrane is placed in a small tube andimmersed in high-salt buffer (1M NaCl; 0.1 mM EDTA; and 20 mM Tris-HCl,pH=8) and then incubated at 65° C. for one hour to remove the DNA fromthe DEAE paper. After the 65° C. incubation, the incubation buffer iscollected and the membrane rinsed with high-salt buffer. The high-saltrinse solution is pooled with the high-salt incubation buffer.

The volume of the high salt-DNA solution is adjusted so that the NaClconcentration is 0.25M, and then three volumes of cold, absolute ethanolare added to the solution. The resulting solution is mixed and placed at−70° C. for 10-20 minutes. The solution is then centrifuged at 15,000rpm for 15 minutes. After another precipitation to remove residual salt,the DNA pellet is rinsed with ethanol, dried, resuspended in 20 μl of TEbuffer, and constitutes about 3 μg of the desired restriction fragmentof Ad2. The purified fragment obtained is dissolved in 10 μl of TEbuffer.

About 6 μl of H₂O and 2 μl of 10× AccI buffer (60 mM NaCl; 60 mMTris-HCl, pH=7.5; 60 mM MgCl₂; 60 mM DTT; and 1 mg/ml BSA) are added tothe solution of the ˜2.4 kb BalI restriction fragment of Ad2. After theaddition of about 2 μl (˜10 units) of restriction enzyme AccI to thesolution of DNA, the reaction is incubated at 37° C. for 2 hours. Afterthe AccI digestion, the DNA is collected by ethanol precipitation andresuspended in 16 μl of H₂O and 2 μl of 10× PvuII buffer (600 mM NaCl;60 mM Tris-HCl, pH=7.5; 60 mM MgCl₂; 60 mM DTT; and 1 mg/ml BSA). Afterthe addition of about 2 μl (about 10 units) of restriction enzyme PvuIIto the solution of DNA, the reaction is incubated at 37° C. for 2 hours.

The AccI-PvuII-digested, ˜2.4 kb BalI restriction fragment of Ad2 isloaded onto an ˜6% polyacrylamide gel and electrophoresed until the˜0.32 kb AccI-PvuII restriction fragment that comprises the Ad2 latepromoter is separated from the other digestion products. The gel isstained with ethidium bromide and viewed using UV light, and the segmentof gel containing the ˜0.32 kb AccI-PvuII restriction fragment is cutfrom the gel, crushed, and soaked overnight at room temperature in ˜250μl of extraction buffer (500 mM NH₄OAc; 10 mM MgOAc; 1 mM EDTA; and 0.1%SDS). The following morning, the mixture is centrifuged, and the pelletis discarded. The DNA in the supernatant is precipitated with ethanol;about 2 μg of tRNA are added to ensure complete precipitation of thedesired fragment. About 0.2 μg of the ˜0.32 kb AccI-PvuII restrictionfragment are obtained and suspended in 7 μl of H₂O.

About 0.25 μg (in 0.5 μl) of BclI linkers (5′-CTGATCAG-3′, availablefrom New England Biolabs), which had been kinased in substantialaccordance with the procedure described in Example 10A, below, was addedto the solution of the ˜0.32 kb AccI-PvuII restriction fragment, andthen, 1 μl (˜1000 units) of T4 DNA ligase and 1 μl of 10× ligase bufferwere added to the solution of DNA, and the resulting reaction wasincubated at 16° C. overnight. The BclI linkers could only ligate to thePvuII end of the AccI-PvuII restriction fragment. DNA sequencing laterrevealed that four BclI linkers attached to the PvuII end of theAccI-PvuII restriction fragment. These extra BclI linkers can be removedby BclI digestion and religation; however, the extra BclI linkers werenot removed as the linkers do not interfere with the proper functioningof the vectors that comprise the extra linkers.

E. coli K12 HB101/pSV2cat cells are obtained in lyophilized form fromthe ATCC under the accession number ATCC 37155, and plasmid pSV2cat DNAwas isolated from the cells in substantial accordance with the procedureof Example 3. A restriction site and function map of plasmid pSV2cat ispresented in FIG. 4 of the accompanying drawings. About 1 mg of plasmidpSV2cat DNA is obtained and dissolved in 1 ml of TE buffer. About 3 μg(3 μl) of the plasmid pSV2cat DNA were added to 2 μl of 10× AccI bufferand 16 μl of H₂O, and then, 3 μl (about 9 units) of restriction enzymeAccI were added to the solution of pSV2cat DNA, and the resultingreaction was incubated at 37° C. for 2 hours. The AccI-digested plasmidpSV2cat DNA was then digested with restriction enzyme StuI by adding 3μl of 10× StuI buffer (1.0M NaCl; 100 mM Tris-HCl, pH=8.0; 100 mM MgCl₂;60 mM DTT; and 1 mg/ml BSA), 5 μl of H₂O, and about 2 μl (about 10units) of restriction enzyme StuI. The resulting reaction was incubatedat 37° C. for 2 hours. The reaction was terminated by extracting thereaction mixture once with phenol, then twice with chloroform. About 0.5μg of the desired fragment was obtained and dissolved in 20 μl of TEbuffer.

About 4 μl of the AccI-StuI-digested plasmid pSV2cat DNA were mixed withabout 7 μl of the ˜0.32 kb AccI-PvuII (with BclI linkers attached)restriction fragment of Ad2, and after the addition of 3 μl of 10×ligase buffer, 15 μl of H₂O, and 2 μl (about 1000 units) of T4 DNAligase, the ligation reaction was incubated at 16° C. overnight. Theligated DNA constituted the desired plasmid pLPcat, a plasmid thatcomprises the Ad2 late promoter positioned so as to drive transcription,and thus expression, of the chloramphenicol acetyltransferase gene. Arestriction site and function map of plasmid pLPcat is presented in FIG.5 of the accompanying drawings.

The ligated DNA was used to transform E. coli K12 HB101 cells insubstantial accordance with the procedure of Example 3. The transformedcells were plated on L agar containing 50 μg/ml ampicillin; restrictionenzyme analysis of plasmid DNA was used to identify the E. coli K12HB101/pLPcat transformants. Plasmid pLPcat DNA was isolated from thetransformants for use in subsequent constructions in substantialaccordance with the plasmid isolation procedure described in Example 3.

B. Final Construction of Plasmid pBLcat

About 88 μg of plasmid pBKneo1 DNA in 50 μl of TE buffer were added to7.5 μl of 10× AccI buffer, 30 μl of H₂O, and 15 μl (about 75 units) ofrestriction enzyme AccI, and the resulting reaction was incubated at 37°C. for 2 hours. The AccI-digested BK virus DNA was loaded on an agarosegel, and the ˜1.4 kb fragment that contains the BK enhancer wasseparated from the other digestion products. The ˜1.4 kb AccIrestriction fragment was then isolated in substantial accordance withthe procedure described in Example 4A. About 5 μg of the fragment wereresuspended in 5 μl of 10× PvuII buffer, 45 μl of H₂O, and 5 μl (about25 units) of restriction enzyme PvuII, and the resulting reaction wasincubated at 37° C. for 2 hours. The PvuII-digested DNA was thenisolated and prepared for ligation in substantial accordance with theprocedure of Example 4A. About 2 μg of the desired ˜1.28 kb AccI-PvuIIfragment were obtained and dissolved in 5 μl of TE buffer.

About 1 μg of plasmid pLPcat DNA was dissolved in 5 μl of 10× AccIbuffer and 40 μl of H₂O. About 5 μl (˜25 units) of restriction enzymeAccI were added to the solution of plasmid pLPcat DNA, and the resultingreaction was incubated at 37° C. The AccI-digested plasmid pLPcat DNAwas precipitated with ethanol and resuspended in 5 μl of 10× StuIbuffer, 40 μl of H₂O, and 5 μl (about 25 units) of restriction enzymeStuI, and the resulting reaction was incubated at 37° C. for 2 hours.The AccI-StuI-digested plasmid pLPcat DNA was precipitated with ethanolseveral times to purify the ˜4.81 kb AccI-StuI restriction fragment thatcomprises the E. coli origin of replication and Ad2 late promoter awayfrom the other digestion product, a restriction fragment about 16 bp insize. About 1 μg of the desired ˜4.81 kb restriction fragment wasobtained and dissolved in 20 μl of TE buffer.

The 5 μl of ˜4.81 kb AccI-StuI restriction fragment of plasmid pLPcatwere added to 5 μl of ˜1.28 kb AccI-PvuII restriction fragment of BKvirus. After the addition of 3 μl of 10× ligase buffer, 15 μl of H₂O,and 2 μl (about 1000 units) of T4 DNA ligase to the mixture of DNA, theresulting ligation reaction was incubated at 16° C. overnight. Theligated DNA constituted the desired plasmid pBLcat. A restriction siteand function map of plasmid pBLcat is presented in FIG. 6 of theaccompanying drawings.

The ligated DNA was used to transform E. coli K12 HB101 cells insubstantial accordance with the procedure described in Example 3. E.coli K12 HB101/pBLcat transformants were identified by restrictionenzyme analysis of their plasmid DNA. Plasmid pBLcat DNA was preparedfor use in subsequent constructions in substantial accordance with theprocedure of Example 3.

EXAMPLE 5 Construction of Plasmid pSBLcat

About 100 μg of plasmid pBLcat DNA were dissolved in 10 μl of 10×HindIII buffer (0.5M NaCl; 0.1M Tris-HCl, pH=8.0; 0.1M MgCl₂; and 1mg/ml BSA) and 80 μl of H₂O. About 10 μl (about 100 units) ofrestriction enzyme HindIII were added to the solution of plasmid pBLcatDNA, and the resulting reaction was incubated at 37° C. for 2 hours. TheHindIII-digested plasmid pBLcat DNA was loaded onto an agarose gel andelectrophoresed until the ˜0.87 kb HindIII restriction fragment thatcomprises the BK enhancer and Ad2 late promoter was well separated fromthe other digestion products; then, the ˜0.87 kb fragment was isolatedand prepared for ligation in substantial accordance with the procedureof Example 4A. About 10 μg of the desired fragment were obtained anddissolved in 50 μl of TE buffer.

About 1 μg of plasmid pSV2cat DNA in 1 μl of TE buffer was dissolved in2 μl of 10× HindIII buffer and 16 μl of H₂O. About 1 μl (about 10 units)of restriction enzyme HindIII was added to the solution of DNA, and theresulting reaction was incubated at 37° C. for 2 hours. The reaction wasstopped by extracting the reaction mixture first with phenol, then twicewith chloroform. The HindIII-digested plasmid pSV2cat DNA wasprecipitated with ethanol and resuspended in 100 μl of TE buffer. TheHindIII-digested plasmid pSV2cat DNA was treated with calf-intestinalalkaline phosphatase in substantial accordance with the procedure ofExample 2 and then resuspended in 10 μl of TE buffer.

About 5 μl of the ˜0.87 kb HindIII restriction fragment of plasmidpBLcat were added to the 10 μl of HindIII-digested plasmid pSV2cat, andthen, 3 μl of 10× ligase buffer, 2 μl (about 1000 units) of T4 DNAligase, and 13 μl of H₂O were added to the solution of DNA, and theresulting reaction was incubated at 16° C. for 2 hours. The ligated DNAconstituted the desired plasmid pSBLcat. The ligated DNA was used totransform E. coli K12 HB101 in substantial accordance with the procedureof Example 3. The transformed cells were plated on L agar containingampicillin, and the plasmid DNA of the ampicillin-resistanttransformants was examined by restriction enzyme analysis to identifythe E. coli K12 HB101/pSBLcat transformants. The ˜0.87 kb HindIIIrestriction fragment that encodes the BK enhancer and Ad2 late promotercould insert into HindIII-digested plasmid pSBLcat in one of twoorientations, only one of which yields plasmid pSBLcat. A restrictionsite and function map of plasmid pSBLcat is presented in FIG. 8 of theaccompanying drawings.

EXAMPLE 6 Construction of Plasmid pL133

A. Construction of Intermediate Plasmid pSV2-HPC8

Plasmid pHC7 comprises a DNA sequence that encodes human protein C. Oneliter of L-broth containing 15 μg/ml tetracycline was inoculated with aculture of E. coli K12 RR1/pHC7 (NRRL B-15926), and plasmid pHC7 DNA wasisolated and purified in substantial accordance with the procedure ofExample 3. About 1 mg of plasmid pHC7 DNA was obtained by thisprocedure, suspended in 1 ml of TE buffer, and stored at −20° C. Arestriction site and function map of plasmid pHC7 is presented in FIG. 9of the accompanying drawings.

Fifty μl of the plasmid pHC7 DNA were mixed with 5 μl (˜50 units) ofrestriction enzyme BanI, 10 μl of 10× BanI reaction buffer (1.5M NaCl;60 mM Tris-HCl, pH=7.9; 60 mM MgCl₂; and 1 mg/ml BSA), and 35 μl of H₂Oand incubated until the digestion was complete. The BanI-digestedplasmid pHC7 DNA was then electrophoresed on a 3.5% polyacrylamide gel(29:1, acrylamide:bis-acrylamide), until the ˜1.25 kb BanI restrictionfragment was separated from the other digestion products.

The region of the gel containing the ˜1.25 kb BanI restriction fragmentwas cut from the gel, placed in a test tube, and broken into smallfragments. One ml of extraction buffer (500 mM NH₄OAc, 10 mM MgOAc, 1 mMEDTA, 1% SDS, and 10 mg/ml tRNA) was added to the tube containing thefragments, and the tube was placed at 37° C. overnight. Centrifugationwas used to pellet the debris, and the supernatant was-transferred to anew tube. The debris was washed once with 200 μl of extraction buffer;the wash supernatant was combined with the first supernatant from theovernight extraction. After passing the supernatant through a plug ofglass wool, two volumes of ethanol were added to and mixed with thesupernatant. The resulting solution was placed in a dry ice-ethanol bathfor ˜10 minutes, and then, the DNA was pelleted by centrifugation.

Approximately 8 μg of the ˜1.25 kb BanI restriction fragment wereobtained by this procedure. The purified fragment was suspended in 10 μlof TE buffer and stored at −20° C. The BanI restriction fragment had tobe modified by the addition of a linker to construct plasmid pSV2-HPC8.The DNA fragments used in the construction of the linker weresynthesized either by using a Systec 1450A DNA Synthesizer (Systec Inc.,3816 Chandler Drive, Minneapolis, Min.) or an ABS 380A DNA Synthesizer(Applied Biosystems, Inc., 850 Lincoln Centre Drive, Foster City, Calif.94404). Many DNA synthesizing instruments are known in the art and canbe used to make the fragments. In addition, the fragments can also beconventionally prepared in substantial accordance with the procedures ofItakura et al., 1977, Science, 198:1056 and Crea et al., 1978, Proc.Nat. Acad. Sci. USA, 75:5765.

Five hundred picomoles of each single strand of the linker were kinasedin 20 μl of reaction buffer, which contained 15 units (˜0.5 μl) T4polynucleotide kinase, 2 μl 10× ligase buffer, 10 μl of 500 μM ATP, and7.5 μl of H₂O. The kinase reaction was incubated at 37° C. for 30minutes, and the reaction was terminated by incubation at 100° C. for 10minutes. In order to ensure complete kination, the reaction was chilledon ice, 2 μl of 0.2M dithiothreitol, 2.5 μl of 5 mM ATP, and 15 units ofT4 polynucleotide kinase were added to the reaction mixture and mixed,and the reaction mixture was incubated another 30 minutes at 37° C. Thereaction was stopped by another 10 minute incubation at 100° C. and thenchilled on ice.

Although kinased separately, the two single strands of the DNA linkerwere mixed together after the kinase reaction. To anneal the strands,the kinase reaction mixture was incubated at 100° C. for 10 minutes in awater bath containing ˜150 ml of water. After this incubation, the waterbath was shut off and allowed to cool to room temperature, a processtaking about 3 hours. The water bath, still containing the tube ofkinased DNA, was then incubated at 4° C. overnight. This processannealed the single strands. The linker constructed had the followingstructure:

The linker was stored at −20° C. until use.

The ˜8 μg of ˜1.25 kb BanI fragment were added to and mixed with the ˜50μl of linker (˜500 picomoles), 1 μl of T4 DNA ligase (˜500 units), 10 μlof 10× ligase buffer, and 29 μl of H₂O, and the resulting ligationreaction was incubated at 4° C. overnight. The ligation reaction wasstopped by a 10 minute incubation at 65° C. The DNA was pelleted byadding NaOAc to a final concentration of 0.3M, adding 2 volumes ofethanol, chilling in a dry ice-ethanol bath, and then centrifuging thesolution.

The DNA pellet was dissolved in 10 μl of 10× ApaI reaction buffer (60 mMNaCl; 60 mM Tris-HCl, pH=7.4; 60 mM MgCl₂; and 60 mM 2-mercaptoethanol),5 μl (˜50 units) of restriction enzyme ApaI, and 85 μl of H₂O, and thereaction was placed at 37° C. for two hours. The reaction was thenstopped and the DNA pelleted as above. The DNA pellet was dissolved in10 μl of 10× HindIII reaction buffer, 5 μl (˜50 units) of restrictionenzyme HindIII, and 85 μl of H₂O, and the reaction was placed at 37° C.for two hours. After the HindIII digestion, the reaction mixture wasloaded onto a 3.5% polyacrylamide gel, and the desired ˜1.23 kbHindIII-ApaI restriction fragment was isolated in substantial accordancewith the procedure described in Example 4A. Approximately 5 μg of thedesired fragment were obtained, suspended in 10 μl of TE buffer, andstored at −20° C.

Fifty μl of plasmid pHC7 DNA were mixed with 5 μl (˜50 units) ofrestriction enzyme PstI, 10 μl of 10× PstI reaction buffer (1.0M NaCl;100 mM Tris-HCl, pH=7.5; 100 mM MgCl₂; and 1 mg/ml BSA), and 35 μl ofH₂O and incubated at 37° C. for two hours. The PstI-digested plasmidpHC7 DNA was then electrophoresed on a 3.5% polyacrylamide gel, and thedesired ˜0.88 kb fragment was purified in substantial accordance withthe procedure described above. Approximately 5 μg of the desiredfragment were obtained, suspended in 10 μl of TE buffer, and stored at−20° C.

The ˜5 μg of ˜0.88 kb PstI fragment were added to and mixed with ˜50 μlof the following linker, which was constructed on an automated DNAsynthesizer:

About 1 μl of T4 DNA ligase (˜10 units), 10 μl 10× ligase buffer, and 29μl H₂O were added to the mixture of DNA, and the resulting ligationreaction was incubated at 4° C. overnight.

The ligation reaction was stopped by a 10 minute incubation at 65° C.After precipitation of the ligated DNA, the DNA pellet was dissolved in10 μl of 10× ApaI reaction buffer, 5 μl (˜50 units) of restrictionenzyme ApaI, and 85 μl of H₂O, and the reaction was placed at 37° C. fortwo hours. The reaction was then stopped and the DNA pelleted onceagain. The DNA pellet was dissolved in 10 μl 10× BglII reaction buffer(1M NaCl; 100 mM Tris-HCl, pH=7.4; 100 mM MgCl₂; 100 mM2-mercaptoethanol; and 1 mg/ml BSA), 5 μl (˜50 units) of restrictionenzyme BgII, and 85 μl H₂O, and the reaction was placed at 37° C. fortwo hours. After the BglII digestion, the reaction mixture was loadedonto a 3.5% polyacrylamide gel, and the desired ˜0.19 kb ApaI-BglIIrestriction fragment was isolated in substantial accordance with theprocedure described above. Approximately 1 μg of the desired fragmentwas obtained, suspended in 10 μl of TE buffer, and stored at −20° C.

Approximately 10 μg of plasmid pSV2gpt DNA (ATCC 37145) were dissolvedin 10 μl of 10× HindIII reaction buffer, 5 μl (˜50 units) of restrictionenzyme HindIII, and 85 μl of H₂O, and the reaction was placed at 37° C.for 2 hours. The reaction mixture was then made 0.25M in NaOAc, andafter the addition of two volumes of ethanol and incubation in a dryice-ethanol bath, the DNA was pelleted by centrifugation. The DNA pelletwas dissolved in 10 μl of 10× BglII buffer, 5 μl (˜50 units) ofrestriction enzyme BglII, and 85 μl of H₂O, and the reaction was placedat 37° C. for two hours. After the BglII digestion, the reaction mixturewas loaded onto a 1% agarose gel, and the fragments were separated byelectrophoresis. The gel was stained with ethidium bromide and viewedunder ultraviolet light, and the band containing the desired ˜5.1 kbHindIII-BglII fragment was cut from the gel and placed in dialysistubing, and electrophoresis was continued until the DNA was out of theagarose. The buffer containing the DNA from the dialysis tubing wasextracted with phenol and CHCl₃, and then, the DNA was precipitated. Thepellet was resuspended in 10 μl of TE buffer and constituted ˜5 μg ofthe desired ˜5.1 kb HindIII-BglII restriction fragment of plasmidpSV2gpt.

Two μl of the ˜1.23 kb HindIII-ApaI restriction fragment, 3 μl of the˜0.19 kb ApaI-BglII fragment, and 2 μl of the ˜5.1 kb HindIII-BglIIfragment were mixed together and then incubated with 10 μl of 10× ligasebuffer, 1 μl of T4 DNA ligase (˜500 units), and 82 μl of H₂O at 16° C.overnight. The ligated DNA constituted the desired plasmid pSV2-HPC8; arestriction site and function map of the plasmid is presented in FIG. 9of the accompanying drawings.

E. coli K12 RR1 (NRRL B-15210) cells were made competent fortransformation in substantial accordance with the procedure described inExample 2. The ligated DNA prepared above was used to transform thecells, and aliguots of the transformation mix were plated on L-agarplates containing 100 μg/ml ampicillin. The plates were then incubatedat 37° C. E. coli K12 RR1/pSV2-HPC8 transformants were verified byrestriction enzyme analysis of their plasmid DNA.

B. Final Construction of Plasmid pL133

Fifty μg of plasmid pSV2-HPC8 were dissolved in 10 μl of 10× HindIIIreaction buffer, 5 μl (˜50 units) of restriction enzyme HindIII, and 85μl of H₂O, and the reaction was incubated at 37° C. for two hours. Afterthe HindIII digestion, the DNA was precipitated, and the DNA pellet wasdissolved in 10 μl 10× SalI reaction buffer (1.5M NaCl; 60 mM Tris-HCl,pH=7.9; 60 mM MgCl₂; 60 mM 2-mercaptoethanol; and 1 mg/ml BSA), 5 μl(˜50 units) of restriction enzyme SalI, and 85 μl of H₂O. The resultingSalI reaction mixture was incubated for 2 hours at 37° C. TheHindIII-SalI-digested plasmid pSV2-HPC8 was loaded onto a 3.5%polyacrylamide gel and electrophoresed until the desired ˜0.29 kbHindIII-SalI restriction fragment was separated from the other reactionproducts. The desired fragment was isolated from the gel; about 2 μg ofthe fragment were obtained and suspended in 10 μl of TE buffer.

Fifty μg of plasmid pSV2-HPC8 were dissolved in 10 μl of 10× BglIIreaction buffer, 5 μl (50 units) of restriction enzyme BglII, and 85 μlof H₂O, and the reaction was incubated at 37° C. for two hours. Afterthe BglII digestion, the DNA was precipitated, and the DNA pellet wasdissolved in 10 μl of 10× SalI reaction buffer, 5 μl (˜50 units) ofrestriction enzyme SalI, and 85 μl of H₂O. The resulting SalI reactionmixture was incubated for 2 hours at 37° C. The SalI-BglII-digestedplasmid pSV2-HPC8 was loaded onto a 3.5% polyacrylamide gel andelectrophoresed until the desired ˜1.15 kb SalI-BglII restrictionfragment was separated from the other reaction products. The ˜1.15 kbSalI-BglII restriction fragment was isolated from the gel; about 8 μg offragment were obtained and suspended in 10 μl of TE buffer.

Approximately 10 μg of plasmid pSV2-β-globin DNA (NRRL B-15928) weredissolved in 10 μl of 10× HindIII reaction buffer, 5 μl (˜50 units) ofrestriction enzyme HindIII, and 85 μl or H₂O, and the reaction wasplaced at 37° C. for 2 hours. The reaction mixture was then made 0.25Min NaOAc, and after the addition of two volumes of ethanol andincubation in a dry ice-ethanol bath, the DNA was pelleted bycentrifugation. The HindIII-digested plasmid pSV2-β-globin was dissolvedin 10 μl of 10× BglII buffer, 5 μl (˜50 units) of restriction enzymeBgII, and 85 μl of H₂O, and the reaction was placed at 37° C. for twohours. After the BglII digestion, the reaction mixture was loaded onto a1% agarose gel, and the fragments were separated by electrophoresis. Thedesired ˜4.2 kb HindIII-BglII restriction fragment was isolated from thegel; about 5 μg of the desired fragment were obtained and suspended in10 μl of TE buffer.

Two μl of the ˜0.29 kb HindIII-SalI fragment of plasmid pSV2-HPC8, 2 μlof the ˜1.15 kb SalI-BglII fragment of plasmid pSV2-HPC8, and 2 μl ofthe ˜4.2 kb HindIII-BglII fragment of plasmid pSV2-β-globin were mixedtogether and ligated in substantial accordance with the procedure ofExample 6A. The ligated DNA constituted the desired plasmid pL133; arestriction site and function map of plasmid pL133 is presented in FIG.9 of the accompanying drawings. The desired E. coli K12 RR1/pL133transformants were constructed in substantial accordance with theteaching of Example 6A, with the exception that plasmid pL133, ratherthan plasmid pSV2-HPC8, was used as the transforming DNA.

EXAMPLE 7 Construction of Plasmid pLPC

About 20 μg of plasmid pBLcat DNA were dissolved in 10 μl of 10× HindIIIbuffer and 80 μl of H₂O. About 10 μl (˜100 units) of restriction enzymeHindIII were added to the solution of plasmid pBLcat DNA, and theresulting reaction was incubated at 37° C. for 2 hours. TheHindIII-digested plasmid pBLcat DNA was loaded onto an agarose gel andelectrophoresed until the ˜0.87 kb HindIII restriction fragment thatcomprises the BK enhancer and Ad2 late promoter was separated from theother digestion products; then, the ˜0.87 kb fragment was isolated andprepared for ligation in substantial accordance with the procedure ofExample 4A. About 2 μg of the desired fragment were obtained anddissolved in 5 μl of TE buffer.

About 1.5 μg of plasmid pL133 DNA was dissolved in 2 μl of 10× HindIIIbuffer and 16 μl of H₂O. About 1 μl (˜10 units) of restriction enzymeHindIII was added to the solution of DNA, and the resulting reaction wasincubated at 37° C. for 2 hours. The DNA was then diluted to 100 μl withTE buffer and treated with calf-intestinal alkaline phosphatase insubstantial accordance with the procedure in Example 2. TheHindIII-digested plasmid pL133 DNA was extracted twice with phenol andonce with chloroform, precipitated with ethanol, and resuspended in 10μl of TE buffer.

About 5 μl of the ˜0.87 kb HindIII restriction fragment of plasmidpBLcat were added to the 1.5 μl of HindIII-digested plasmid pL133, andthen, 1 μl of 10× ligase buffer, 1 μl (˜1000 units) of T4 DNA ligase,and 1.5 μl of H₂O were added to the solution of DNA, and the resultingreaction was incubated at 16° C. overnight. The ligated DNA constitutedthe desired plasmid pLPC. A restriction site and function map of plasmidpLPC is presented in FIG. 10 of the accompanying drawings.

The ligated DNA was used to transform E. coli K12 HB101 in substantialaccordance with the procedure of Example 3. The transformed cells wereplated on L agar containing ampicillin, and the plasmid DNA of theampicillin-resistant transformants was examined by restriction enzymeanalysis to identify the E. coli K12 HB101/pLPC transformants. The ˜0.87kb HindIII restriction fragment that encodes the BK enhancer and Ad2late promoter could insert into HindIII-digested plasmid pL133 in one oftwo orientations, only one of which yields plasmid pLPC.

EXAMPLE 8 Construction of Plasmids pLPC4 and pLPC5

About 1 μg (1 μl) of the BK virus DNA prepared in Example 1 and 1 μg ofplasmid pLPC (1 μl) were dissolved in 2 μl of 10× EcoRI buffer and 14 μlof H₂O. About 2 μl (˜10 units) of restriction enzyme EcoRI were added tothe solution of DNA, and the resulting reaction was incubated at 37° C.for 2 hours. The EcoRI-digested mixture of BK virus and plasmid pLPC DNAwas extracted once with buffered phenol and once with chloroform. Then,the DNA was collected by adjusting the NaCl concentration to 0.25M,adding two volumes of ethanol, incubating the solution in a dryice-ethanol bath for 2 minutes, and centrifuging the solution to pelletthe DNA. The supernatant was discarded, and the DNA pellet was rinsedwith 70% ethanol, dried, and resuspended in 12 μl of TE buffer.

About 13 μl of H₂O and 3 μl of 10× ligase buffer were added to theEcoRI-digested mixture of BK virus and plasmid pLPC DNA. Two μl (˜1000units) of T4 DNA ligase were added to the solution of DNA, and theresulting reaction was incubated at 16° C. for 2 hours. The ligated DNAconstituted the desired plasmids pLPC4 and pLPC5, which differ only withrespect to the orientation of the inserted BK virus DNA. A restrictionsite and function map of plasmid pLPC4 is presented in FIG. 11 of theaccompanying drawings.

The ligated DNA constituted the desired plasmids pLPC4 and pLPC5 and wasused to transform E. coli K12 HB101 competent cells in substantialaccordance with the procedure of Example 3. The transformed cells wereplated on L agar containing 100 μg/ml ampicillin. The E. coli K12HB101/pLPC4 and E. coli K12 HB101/pLPC5 transformants were identified bytheir ampicillin-resistant phenotype and by restriction enzyme analysisof their plasmid DNA.

EXAMPLE 9 Construction of Plasmids pLPChyg1 and pLPChyg2

E. coli K12 RR1/pSV2hyg cells are obtained from the Northern RegionalResearch Laboratory under the accession number NRRL B-18039. PlasmidpSV2hyg DNA is obtained from the cells in substantial accordance withthe procedure of Example 3. A restriction site and function map ofplasmid pSV2hyg is presented in FIG. 12 of the accompanying drawings.

About 10 μg (in 10 μl of TE buffer) of plasmid pSV2hyg were added to 2μl of 10× BamHI buffer and 6 μl of H₂O. About 2 μl (about 20 units) ofrestriction enzyme BamHI were added to the solution of DNA, and theresulting reaction was incubated at 37° C. for 2 hours. The reaction wasextracted first with phenol and then was extracted twice withchloroform. The BamHI-digested plasmid pSV2hyg DNA was loaded onto anagarose gel, and the hygromycin resistance gene-containing, ˜2.5 kbrestriction fragment was isolated in substantial accordance with theprocedure described in Example 4A.

About 5 μl of 10× Klenow buffer (0.2 mM in each of the four dNTPs; 0.5MTris-HCl, pH=7.8; 50 mM MgCl₂; 0.1M 2-mercaptoethanol; and 100 μg/mlBSA) and 35 μl of H₂O were added to the solution of BamHI-digestedplasmid pSV2hyg DNA, and then, about 25 units of Klenow enzyme (about 5μl, as marketed by BRL) were added to the mixture of DNA, and theresulting reaction was incubated at 16° C. for 30 minutes. TheKlenow-treated, BamHI-digested plasmid pSV2hyg DNA was extracted oncewith phenol and once with chloroform and then precipitated with ethanol.About 2 μg of the desired fragment were obtained and suspended in 5 μlof TE buffer.

About 10 μg (10 μl) of plasmid pLPC DNA were added to 2 μl of 10× StuIbuffer and 6 μl of H₂O. About 2 μl (˜10 units) of restriction enzymeStuI were added to the solution of DNA, and the resulting reaction wasincubated at 37° C. for 2 hours. The StuI-digested plasmid pLPC DNA wasprecipitated with ethanol, collected by centrifugation, and resuspendedin 2 μl of 10× NdeI buffer (1.5M NaCl; 0.1M Tris-HCl, pH=7.8; 70 mMMgCl₂; 60 mM 2-mercaptoethanol; and 1 mg/ml BSA) and 16 μl of H₂O. About2 μl (˜10 units) of restriction enzyme NdeI were added to the solutionof StuI-digested DNA, and the resulting reaction was incubated at 37° C.for 2 hours.

The NdeI-StuI-digested plasmid pLPC DNA was precipitated with ethanol,collected by centrifugation, and resuspended in 5 μl of 10× Klenowbuffer and 40 μl of H₂O. About 5 μl (˜25 units) of Klenow enzyme wereadded to the solution of DNA, and the resulting reaction was incubatedat 16° C. for 30 minutes. After the Klenow reaction, the reactionmixture was loaded onto an agarose gel, and the ˜5.82 kb NdeI-StuIrestriction fragment was isolated from the gel. About 5 μg of thedesired fragment were obtained and suspended in 5 μl of TE buffer.

About 2 μl of the ˜2.5 kb Klenow-treated BamHI restriction fragment ofplasmid pSV2hyg were mixed with about 1 μl of the ˜5.82 kbKlenow-treated NdeI-StuI restriction fragment of plasmid pLPC, and about3 μl of 10× ligase buffer, 2 μl of T4 DNA ligase (˜1000 units), 1 μl ofT4 RNA ligase (˜1 unit), and 14 μl of H₂O were added to the solution ofDNA. The resulting reaction was incubated at 16° C. overnight. Theligated DNA constituted the desired plasmids pLPChyg1 and pLPChyg2,which differ only with respect to the orientation of the ˜2.5 kbKlenow-treated, BamHI restriction fragment of plasmid pSV2hyg. Arestriction site and function map of plasmid pLPChyg1 is presented inFIG. 13 of the accompanying drawings. The ligated DNA was used totransform E. coli K12 HB101 in substantial accordance with the procedureof Example 3. The desired E. coli K12 HB101/pLPChyg1 and E. coli K12HB101/pLPChyg2 transformants were plated on L agar containing ampicillinand identified by restriction enzyme analysis of their plasmid DNA.

EXAMPLE 10 Construction of Plasmid pBW32

A. Construction of Intermediate Plasmid pTPA103

Plasmid pTPA102 comprises the coding sequence of human tissueplasminogen activator (TPA). Plasmid pTPA102 can be isolated from E.coli K12 MM294/pTPA102, a strain available from the Northern RegionalResearch Laboratory under the accession number NRRL B-15834. Arestriction site and function map of plasmid pTPA102 is presented inFIG. 14 of the accompanying drawings. Plasmid pTPA102 DNA is isolatedfrom E. coli K12 MM294/pTPA102 in substantial accordance with theprocedure of Example 2.

About 50 μg of plasmid pTPA102 (in about 50 μl of TE buffer) were addedto 10 μl of 10× TthlllI buffer (0.5M NaCl; 80 mM Tris-HCl, pH=7.4; 80 mMMgCl₂; 80 mM 2-mercaptoethanol; and 1 mg/ml BSA) and 80 μl of H₂O. About10 μl (˜50 units) of restriction enzyme TthlllI were added to thesolution of DNA, and the resulting reaction was incubated at 65° C. for2 hours. The reaction mixture was loaded onto an agarose gel, and the˜4.4 kb TthlllI restriction fragment that comprises the TPA codingsequence was isolated from the gel. The other digestion products, 3.1 kband 0.5 kb restriction fragments, were discarded. About 10 μg of thedesired ˜4.4 kb TthlllI restriction fragment were obtained and suspendedin 10 μl of TE buffer.

About 5 μl of 10× Klenow buffer and 30 μl of H₂O were added to thesolution comprising the ˜4.4 kb TthlllI restriction fragment, and afterthe further addition of about 5 μl of Klenow enzyme (˜5 units), thereaction mixture was incubated at 16° C. for 30 minutes. After theKlenow reaction, the DNA was precipitated with ethanol and resuspendedin 3 μl of 10× ligase buffer and 14 μl of H₂O.

BamHI linkers (New England Biolabs), which had the following sequence:

were kinased and prepared for ligation by the following procedure. Fourμl of linkers (˜2 μg) were dissolved in 20.15 μl of H₂O and 5 μl of 10×kinase buffer (500 mM Tris-HCl, pH=7.6 and 100 mM MgCl₂), incubated at90° C. for two minutes, and then cooled to room temperature. Five μl ofγ-³²P-ATP (˜20 μCi), 2.5 μl of 1M DTT, and 5 μl of polynucleotide kinase(18 10 units) were added to the mixture, which was then incubated at 37°C. for 30 minutes. Then, 3.35 μl of 0.01M ATP and 5 μl of kinase wereadded, and the reaction was continued for another 30 minutes at 37° C.The radioactive ATP aids in determining whether the linkers have ligatedto the target DNA.

About 10 μl of the kinased BamHI linkers were added to the solution of˜4.4 kb TthlllI restriction fragment, and after the addition of 2 μl ofT4 DNA ligase (˜1000 units) and 1 μl of T4 RNA ligase (˜2 units), theligation reaction was incubated overnight at 4° C. The ligated DNA wasprecipitated with ethanol and resuspended in 5 μl of 10× HindIII bufferand 40 μl of H₂O. About 5 μl (˜50 units) of restriction enzyme HindIIIwere added to the solution of DNA, and the resulting reaction wasincubated at 37° C. for 2 hours.

The HindIII-digested DNA was precipitated with ethanol and resuspendedin 10 μl of 10× BamHI buffer and 90 μl of H₂O. About 10 μl (˜100 units)of restriction enzyme BamHI were added to the solution of DNA, and theresulting reaction was incubated at 37° C. for 2 hours. After the BamHIdigestion, the reaction mixture was loaded onto an agarose gel, and the˜2.0 kb BamHI-HindIII restriction fragment was isolated from the gel.About 4 μg of the desired fragment were obtained and suspended in about5 μl of TE buffer.

To construct plasmid pTPA103, the ˜2.0 kb BamHI-HindIII restrictionfragment derived from plasmid pTPA102 was inserted intoBamHI-HindIII-digested plasmid pRC. Plasmid pRC was constructed byinserting an ˜288 bp EcoRI-ClaI restriction fragment that comprises thepromoter and operator (trpPO) sequences of the E. coli trp operon intoEcoRI-Clal-digested plasmid pKC7. Plasmid pKC7 can be obtained from theAmerican Type Culture Collection in E. coli K12 N100/pKC7 under theaccession number ATCC 37084. The ˜288 bp EcoRI-ClaI restriction fragmentthat comprises the trpPO can be isolated from plasmid pTPA102, which canbe isolated from E. coli K12 MM294/pTPA102 (NRRL B-15834). Plasmid pKC7and plasmid pTPA102 DNA can be obtained from the aforementioned celllines in substantial accordance with the procedure of Example 3. This˜0.29 kb EcoRI-ClaI restriction fragment of plasmid pTPA102 comprisesthe transcription activating sequence and most of the translationactivating sequence of the E. coli trp gene and has the sequencedepicted below:

Thus, to construct plasmid pRC, about 2 μg of plasmid pKC7 in 10 μl ofTE buffer were added to 2 μl of 10× ClaI buffer (0.5M NaCl; 60 mMTris-HCl, pH=7.9, 60 MM MgCl₂; and 1 mg/ml BSA) and 6 μl of H₂O. About 2μl (˜10 units) of restriction enzyme ClaI were added to the solution ofplasmid pKC7 DNA, and the resulting reaction was incubated at 37° C. for2 hours. The ClaI-digested plasmid pKC7 DNA was precipitated withethanol and resuspended in 2 μl of 10× EcoRI buffer and 16 μl of H₂O.About 2 μl (˜10 units) of restriction enzyme EcoRI were added to thesolution of ClaI-digested plasmid pKC7 DNA, and the resulting reactionwas incubated at 37° C. for 2 hours.

The EcoRI-ClaI-digested plasmid pKC7 DNA was extracted once with phenoland then twice with chloroform. The DNA was then precipitated withethanol and resuspended in 3 μl of 10× ligase buffer and 20 μl of H₂O. Arestriction site and function map of plasmid pKC7 can be obtained fromManiatis et al., Molecular Cloning (Cold Spring Harbor Laboratory,1982), page 8.

About 20 μg of plasmid pTPA102 in about 20 μl of TE buffer were added to10 μl of 10× ClaI buffer and 60 μl of H₂O. About 10 μl (˜50 units) ofrestriction enzyme ClaI were added to the solution of plasmid pTPA102DNA, and the resulting reaction was incubated at 37° C. for 2 hours. TheClaI-digested plasmid pTPA102 DNA was precipitated with ethanol andresuspended in 10 μl of 10× EcoRI buffer and 80 μl of H₂O. About 10 μl(˜50 units) of restriction enzyme EcoRI were added to the solution ofClaI-digested plasmid pTPA102 DNA, and the resulting reaction wasincubated at 37° C. for 2 hours.

The EcoRI-ClaI-digested plasmid pTPA102 DNA was extracted once withphenol, loaded onto a 7% polyacrylamide gel, and electrophoresed untilthe ˜288 bp EcoRI-ClaI restriction fragment that comprises the trpPO wasseparated from the other digestion products. The ˜288 bp EcoRI-ClaIrestriction fragment was isolated from the gel; about 1 μg of thedesired fragment was obtained, suspended in 5 μl of TE buffer, and addedto the solution of EcoRI-ClaI-digested plasmid pKC7 DNA prepared asdescribed above. About 2 μl (˜1000 units) of T4 DNA ligase were thenadded to the mixture of DNA, and the resulting ligation reaction wasincubated at 16° C. for 2 hours. The ligated DNA constituted the desiredplasmid pRC DNA.

The ligated DNA was used to transform E. coli K12 HB101 competent cellsin substantial accordance with the procedure of Example 2. Thetransformed cells were plated on L agar containing 100 μg/ml ampicillin,and the ampicillin-resistant transformants were screened by restrictionenzyme analysis of their plasmid DNA to identify the desired E. coli K12HB101/pRC colonies. Plasmid pRC DNA was obtained from the E. coli K12HB101/pRC transformants in substantial accordance with the procedure ofExample 3.

About 2 μg of plasmid pRC DNA in 2 μl of TE buffer were added to 2 μl of10× HindIII buffer and 16 μl of H₂O. About 2 μl (−10 units) ofrestriction enzyme HindIII were added to the solution of plasmid pRCDNA, and the resulting reaction was incubated at 37° C. for two hours.The HindIII-digested plasmid pRC DNA was precipitated with ethanol andresuspended in 2 μl of 10× BamHI buffer and 16 μl of H₂O. About 2 μl(−10 units) of restriction enzyme BamHI were added to the solution ofHindIII-digested plasmid pRC DNA, and the resulting reaction wasincubated at 37° C. for 2 hours.

The BamHI-HindIII-digested plasmid pRC DNA was extracted once withphenol and then twice with chloroform. The DNA was precipitated withethanol and resuspended in 3 μl of 10× ligase buffer and 20 μl of H₂O.The ˜4 μg (in ˜5 μl of TE buffer) of ˜2.0 kb HindIII-BamHI restrictionfragment of plasmid pTPA102 were then added to the solution ofBamHI-HindIII-digested plasmid pRC DNA. About 2 μl (˜1000 units) of T4DNA ligase were added to the mixture of DNA, and the resulting reactionwas incubated at 16° C. for 2 hours. The ligated DNA constituted thedesired plasmid pTPA103 DNA.

To reduce undesired transformants, the ligated DNA was digested withrestriction enzyme NcoI, which cuts plasmid pRC but not plasmid pTPA103.Thus, digestion of the ligated DNA with NcoI reduces undesiredtransformants, because linearized DNA transforms E. coli at a lowerfrequency than closed, circular DNA. To digest the ligated DNA, the DNAwas first precipitated with ethanol and then resuspended in 2 μl of 10×NcoI buffer (1.5M NaCl; 60 mM Tris-HCl, pH=7.8; 60 mM MgCl₂; and 1 mg/mlBSA) and 16 μl of H₂O. About 2 μl (˜10 units) of restriction enzyme NcoIwere added to the solution of DNA, and the resulting reaction wasincubated at 37° C. for 2 hours.

The ligated and then NcoI-digested DNA was used to transform E. coli K12RV308 (NRRL B-15624). E. coli K12 RV308 cells were made competent andtransformed in substantial accordance with the procedure of Example 3.The transformation mixture was plated on L agar containing 100 μg/mlampicillin. The ampicillin-resistant transformants were tested forsensitivity to kanamycin, for though plasmid pRC confers kanamycinresistance, plasmid pTPA103 does not. The ampicillin-resistant,kanamycin-sensitive transformants were then used to prepare plasmid DNA,and the plasmid DNA was examined by restriction enzyme analysis toidentify the E. coli K12 RV308/pTPA103 transformants. A restriction siteand function map of plasmid pTPA103 is presented in FIG. 14 of theaccompanying drawings. Plasmid pTPA103 DNA was isolated from the E. coliK12 RV308/pTPA103 cells in substantial accordance with the procedure ofExample 3.

B. Construction of Intermediate Plasmid pBW25

About 1 μg of plasmid pTPA103 DNA in 1 μl of TE buffer was added to 2 μlof 10× BglII buffer and 16 μl of H₂O. About 1 μl (˜5 units) ofrestriction enzyme BglII was added to the solution of plasmid pTPA103DNA, and the resulting reaction was incubated at 37° C. for 2 hours. TheBglII-digested plasmid pTPA103 DNA was precipitated with ethanol andresuspended in 5 μl of 10× Klenow buffer and 44 μl of H₂O. About 1 μl ofKlenow enzyme (˜1 unit) was added to the solution of BglII-digestedplasmid pTPA103 DNA, and the resulting reaction was incubated at 16° C.for 2 hours. The Klenow-treated, BglII-digested plasmid pTPA103 DNA wasprecipitated with ethanol and resuspended in 3 μl of 10× ligase bufferand 22 μl of H₂O.

About 2 μl (0.2 μg) of unkinased NdeI linkers (New England Biolabs) ofsequence:

were added to the solution of Klenow-treated, BglII-digested plasmidpTPA103 DNA, together with 2 μl (˜1000 units) of T4 DNA ligase and 1 μl(˜2 units) of T4 RNA ligase, and the resulting ligation reaction wasincubated at 4° C. overnight. The ligated DNA constituted plasmidpTPA103derNdeI, which is substantially similar to plasmid pTPA103,except plasmid pTPA103derNdeI has an NdeI recognition sequence whereplasmid pTPA103 has a BglII recognition sequence.

The ligated DNA was used to transform E. coli K12 RV308 competent cellsin substantial accordance with the procedure described in Example 2. Thetransformed cells were plated on L-agar containing ampicillin, and theE. coli K12 RV308/pTPA103derNdeI transformants were identified byrestriction enzyme analysis of their plasmid DNA. Plasmid pTPA103derNdeIDNA was isolated from the transformants for use in subsequentconstructions in substantial accordance with the procedure of Example 3.

About 10 μg of plasmid pTPA103derNdeI DNA in 10 μl of TE buffer wereadded to 2 μl of 10× AvaII buffer (0.6M NaCl; 60 mM Tris-HCl, pH=8.0;0.1M MgCl₂; 60 mM 2-mercaptoethanol; and 1 mg/ml BSA) and 6 μl of H₂O.About 2 μl (˜10 units) of restriction enzyme AvaII were added to theDNA, the resulting reaction was incubated at 37° C. for 2 hours. TheAvaII-digested DNA was loaded onto an agarose gel and electrophoreseduntil the ˜1.4 kb restriction fragment was separated from the otherdigestion products. The ˜1.4 kb AvaII restriction fragment of plasmidpTPA103derNdeI was isolated from the gel; about 2 μg of the desiredfragment were obtained and suspended in 5 μl of TE buffer.

About 5 μl of 10× Klenow buffer, 35 μl of H₂O, and 5 μl (˜5 units) ofKlenow enzyme were added to the solution of ˜1.4 kb AvaII restrictionfragment, and the resulting reaction was incubated at 16° C. for thirtyminutes. The Klenow-treated DNA was precipitated with ethanol andresuspended in 3 μl of 10× ligase buffer and 14 μl of H₂O.

About 2 μg of HpaI linkers of sequence:

were kinased in substantial accordance with the procedure of Example10A. About 10 μl of the kinased linkers were added to the solution ofKlenow-treated, ˜1.4 kb AvaII restriction fragment of plasmidpTPA103derNdeI together with 2 μl (˜1000 units) of T4 DNA ligase and 1μl (˜1 unit) of T4 RNA ligase, and the resulting reaction was incubatedat 16° C. overnight.

The ligated DNA was extracted once with phenol, extracted twice withchloroform, precipitated with ethanol, and resuspended in 2 μl of 10×EcoRI buffer and 16 μl of H₂O. About 2 μl (˜10 units) of restrictionenzyme EcoRI were added to the solution of DNA, and the resultingreaction was incubated at 37° C. for 2 hours. The EcoRI-digested DNA wasextracted once with phenol, extracted twice with chloroform,precipitated with ethanol, and resuspended in 3 μl of 10× ligase bufferand 20 μl of H₂O. The fragment, which is about 770 bp in size andencodes the trpPO and the amino-terminus of TPA, thus prepared had oneEcoRI-compatible end and one blunt end and was ligated intoEcoRI-digested plasmid pUC19 to form plasmid pUC19TPAFE.

About 2 μl of plasmid pUC19 (available from Bethesda ResearchLaboratories) were dissolved in 2 μl of 10× SmaI buffer (0.2M KCl; 60 mMTris-HCl, pH=8.0; 60 mM MgCl₂; 60 mM 2-mercaptoethanol; and 1 mg/ml BSA)and 16 μl of H₂O. About 2 μl (˜10 units) of restriction enzyme SmaI wereadded to the solution of DNA, and the resulting reaction was incubatedat 25° C. for 2 hours. The SmaI-digested plasmid pUC19 DNA wasprecipitated with ethanol, collected by centrifugation, and resuspendedin 2 μl of 10× EcoRI buffer and 16 μl of H₂O. About 2 μl (˜10 units) ofrestriction enzyme EcoRI were added to the solution of SmaI-digestedplasmid pUC19 DNA, and the resulting reaction was incubated at 37° C.for 2 hours. The EcoRI-SmaI-digested plasmid pUC19 DNA was extractedonce with phenol, extracted twice with chloroform, and resuspended in 5μl of TE buffer.

The EcoRI-SmaI-digested plasmid pUC19 DNA was added to the solutioncontaining the ˜770 bp EcoRI-blunt end restriction fragment derived fromplasmid pTPA103derNdeI. About 2 μl (˜1000 units) of T4 DNA ligase wereadded to the mixture of DNA, and the resulting reaction was incubated at16° C. overnight. The ligated DNA constituted the desired plasmidpUC19TPAFE. A restriction site and function map of plasmid pUC19TPAFE ispresented in FIG. 14 of the accompanying drawings.

The multiple-cloning site of plasmid pUC19, which comprises the EcoRIand SmaI recognition sequences utilized in the construction of plasmidpUC19TPAFE, is located within the coding sequence for the lacZ αfragment. Expression of the lacZ α fragment in cells that contain thelacZ ΔM15 mutation, a mutation in the lacZ gene that encodesβ-galactosidase, allows those cells to express a functionalβ-galactosidase molecule and thus allows those cells to hydrolyze X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), a colorlesscompound, to its indigo-colored hydrolysis product. Insertion of DNAinto the multiple-cloning site of plasmid pUC19 interrupts the codingsequence for the lacZ α fragment, and cells with the lacZ ΔM15 mutationthat host such a plasmid are unable to hydrolyze X-Gal (this sameprinciple is utilized when cloning into plasmid pUC8; see Example 2).The ligated DNA that constituted plasmid pUC19TPAFE was used totransform E. coli K12 RR1ΔM15 (NNRL B-15440) cells made competent fortransformation in substantial accordance with the procedure of Example2.

The transformed cells were plated on L agar containing 100 μg/mlampicillin; 40 μg/ml X-Gal; and 1 mM IPTG. Colonies that failed toexhibit the indigo color were subcultured and used to prepare plasmidDNA; the E. coli K12 RR1ΔM15/pUC19TPAFE transformants were identified byrestriction enzyme analysis of their plasmid DNA. Plasmid pUC19TPAFE DNAwas isolated from the E. coli K12 RR1ΔM15/pUC19TPAFE cells for use insubsequent constructions in substantial accordance with the procedure ofExample 3.

About 7 μl of plasmid of pUC19TPAFE in 20 μl of TE buffer were added to10 μl of 10× HpaI buffer (0.2M KCl; 0.1M Tris-HCl, pH=7.4; and 0.1MMgCl₂) and 70 μl of H₂O). About 3 μl (˜6 units) of restriction enzymeHpaI were added to the solution of plasmid pUC19TPAFE DNA, and theresulting reaction was incubated at 37° C. for 20 minutes; the shortreaction period was designed to yield a partial HpaI digest. Thereaction was adjusted to 150 μl of 1× BamHI buffer (150 mM NaCl; 10 mMTris-HCl, pH=8.0; and 10 mM MgCl₂; raising the salt concentrationinactivates HpaI). About 1 μl (˜16 units) of restriction enzyme BamHIwere added to the solution of partially-HpaI-digested DNA, and theresulting reaction was incubated at 37° C. for 90 minutes.

The BamHI-partially-HpaI-digested plasmid pUC19TPAFE DNA wasconcentrated by ethanol precipitation, loaded onto a 1.5% agarose gel,and the ˜3.42 kb HpaI-BamHI restriction fragment that comprises thereplicon, β-lactamase gene, and all of the TPA-encoding DNA of plasmidof pUCATPAFE was isolated from the gel by cutting out the segment of thegel that contained the desired fragment, freezing the segment, and thensqueezing the liquid from the segment. The DNA was precipitated from theliquid by an ethanol precipitation. About 1 μg of the desired fragmentwas obtained and suspended in 20 μl of TE buffer.

About 10 μg of plasmid pTPA103 in 10 μl of TE buffer were dissolved in10 μl of 10× ScaI buffer (1.0M NaCl; 60 mM Tris-HCl, pH=7.4; and 60 mMMgCl₂) 10 mM DTT; and 1 mg/ml BSA) and 80 μl of H₂O. About 3 μl (˜18units) of restriction enzyme ScaI were added to the solution of plasmidpTPA103 DNA, the resulting reaction was incubated at 37° C. for 90minutes. The reaction volume was adjusted to 150 μl of 1× BamHI buffer,and about 1 μl (˜16 units) of restriction enzyme BamHI was added to themixture, which was then incubated at 37 ° C. for 90 minutes. The DNA wasprecipitated with ethanol, collected by centrifugation, and resuspendedin preparation for electrophoresis. The ScaI-BamHI-digested plasmidpTPA103 DNA was loaded onto a 1.5% agarose gel and electrophoresed untilthe ˜1.015 kb ScaI-BamHI restriction fragment was separated from theother digestion products. The ˜1.015 ScaI-BamHI restriction fragmentthat comprises the TPA carboxy-terminus-encoding DNA of plasmid pTPA103was isolated from the gel; about 0.5 μg of the desired fragment wereobtained and dissolved in 20 μl of glass-distilled H₂O.

About 2 μl of the ˜3.42 kb BamHI-HpaI restriction fragment of plasmidpUC19TPAFE were added to 2 μof the ˜1.015 kb ScaI-BamHI restrictionfragment of plasmid pTPA103 together with 2 μl of 10× ligase buffer and1 μl (˜1 Weiss unit; the ligase was obtained from Promega Biotec, 2800S. Fish Hatchery Road, Madison, Wis. 53711) of T4 DNA ligase, and theresulting reaction was incubated at 16° C. overnight. The ligand DNAconstituted the desired plasmid pBW25. A restriction site and functionmap of plasmid pBW25 is presented in FIG. 14 of the accompanyingdrawings.

The ligated DNA was used to transform E. coli K12 JM105 (available fromBRL) that were made competent for transformation in substantialaccordance with the procedure of Example 2, except that 50 mM CaCl₂ wasused in the procedure. The transformed cells were plated on BHI (DifcoLaboratories, Detroit, Mich.) containing 100 μg/ml ampicillin, and theE. coli K12 JM105/pBW25 transformants were identified by restrictionenzyme analysis of their plasmid DNA. Digestion of plasmid pBW25 withrestriction enzyme EcoRI yields ˜3.38 kb and ˜1.08 kb restrictionfragments. Plasmid pBW25 is prepared for use in subsequent constructionsin substantial accordance with the procedure of Example 3.

C. Site-Specific Mutagenesis of the TPA Coding Region and Constructionof Plasmid pBW28

About 5 μg of plasmid pBW25 in 10 μl of glass-distilled H₂O were addedto about 10 μl of 10× HindIII reaction buffer and 80 μl of H₂O. About 1μl (˜20 units) of restriction enzyme HindIII was added to the solutionof plasmid pBW25 DNA, and the resulting reaction was incubated at 37° C.for 90 minutes. About 3 μl (˜24 units) of restriction enzyme EcoRI and10 μl of 1M Tris-HCl, pH=7.6, were added to the solution ofHindIII-digested plasmid pBW25 DNA, and the resulting reaction wasincubated at 37° C. for 90 minutes. The EcoRI-HindIII-digested plasmidpBW25 DNA was concentrated by ethanol precipitation, loaded onto a 1.5%agarose gel, and electrophoresed until the ˜810 bp EcoRI-HindIIIrestriction fragment was separated from the other digestion products.About 0.5 μg of the ˜810 bp EcoRI-HindIII restriction fragment wasisolated from the gel, prepared for ligation, and resuspended in 20 μlof glass-distilled H₂O.

About 4.5 μg of the replicative form (RF) of M13mp8 DNA (available fromNew England Biolabs) in 35 μl of glass-distilled H₂O were added to 10 μlof 10× HindIII buffer and 55 μl of H₂O. About 1 μl (˜20 units) ofrestriction enzyme HindIII was added to the solution of M13mp8 DNA, andthe resulting reaction was incubated at 37° C. for 1 hour. About 3 μl(˜24 units) of restriction enzyme EcoRI and about 10 μl of 1M Tris-HCl,pH=7.6, were added to the solution of HindIII-digestion M13mp8 DNA, andthe resulting reaction was incubated at 37° C. for 1 hour. TheHindIII-EcoRI-digested M13mp8 DNA was collected by ethanolprecipitation, resuspended in preparation for agarose gelelectrophoresis, and the large restriction fragment isolated by gelelectrophoresis. About 1 μg of the large EcoRI-HindIII restrictionfragment of M13mp8 was obtained and suspended in 20 μl ofglass-distilled H₂O. About 2 μl of the large EcoRI-HindIII restrictionfragment of M13mp8, 2 μl of 10× ligase buffer, 12 μl of H₂O and ˜1 μl(˜1 Weiss unit) of T4 DNA ligase were added to 3 μl of the ˜810 bpEcoRI-HindIII restriction fragment of plasmid pBW25, and the resultingligation reaction was incubated at 16° C. overnight.

E. coli JM103 cells, available from BRL, were made competent andtransfected with the ligation mix in substantial accordance with theprocedure described in the BRL M13 Cloning/‘Dideoxy’ SequencingInstruction Manual, except that the amount of DNA used per transfectionwas varied. Recombinant plaques were identified by insertionalinactivation of the β-galactosidase α-fragment-encoding gene, whichresults in the loss of the ability of cleave X-gal to its indigo-coloredcleavage product. For screening purposes, six white plaques were pickedinto 2.5 ml of L broth, to which was added 0.4 ml of E. coli K12 JM103,cultured in minimal media stock to insure retention of the F episomethat carries proAB, in logarithmic growth phase. The plaque-containingsolutions were incubated in an air-shaker at 37° C. for 8 hours. Cellsfrom 1.5 ml aliquots were pelleted and RF DNA isolated in substantialaccordance with the alkaline miniscreen procedure of Birnboim and Doly,1979, Nuc. Acids Res. 7:1513. The remainder of each culture was storedat 4° C. for stock. The desired phage, designated pM8BW26, contained the˜810 bp EcoRI-HindIII restriction fragment of plasmid pBW25 ligated tothe ˜7.2 kb EcoRI-HindIII restriction fragment of M13mp8.

About fifty ml of log phase E. coli JM103 were infected with pM8BW26 andincubated in an air-shaker at 37° C. for 18 hours. The infected cellswere pelleted by low speed centrifugation, and single-stranded pM8BW26DNA was prepared from the culture supernatant by scaling up theprocedure given in the Instruction manual. Single-stranded pM8BW26 wasmutagenized in substantial accordance with the teaching of Adelman etal., 1983, DNA 2(3): 183-193, except that the Klenow reaction was doneat room temperature for 30 minutes, then at 37° C. for 60 minutes, thenat 10° C. for 18 hours. In addition, the S1 treatment was done at 20°C., the salt concentration of the buffer was one-half that recommendedby the manufacturer, and the M13 sequencing primer (BRL) was used. Thesynthetic oligodeoxyribonucleotide primer used to delete the codingsequence for amino acid residues 87 through 261 of native TPA was

 5′-GGGAAGTGCTGTGAAATATCCACCTGCGGCCTGAGA-3′.

The resulting mutagenesis mix was used to transfect E. coli K12 JM103 insubstantial accordance with the infection procedure described above.Desired mutants were identified by restriction enzyme analysis of RF DNAand by Maxam and Gilbert DNA sequencing. The desired mutant, which hadthe coding sequence for amino acid residues 87 through 261 of native TPAdeleted, was designated pM8BW27.

To construct plasmid pBW28, a variety of DNA fragments are needed. Thefirst of these fragments was obtained by adding ˜20 μg of RF pM8BW27 DNAin 20 μl of glass-distilled H₂O to 10 μl of 10× NdeI buffer and 60 μl ofH₂O. About 10 μl (˜50 units) of restriction enzyme NdeI were added tothe mixture of plasmid pM8BW27 DNA, and the resulting reaction wasincubated at 37° C. for two hours. The NdeI-digested plasmid pM8BW27 DNAwas precipitated with ethanol, collected by centrifugation, andresuspended in 10 μl of 10× EcoRI and 90 μl of H₂O. About 10 μl (˜50units) of restriction enzyme EcoRI were added to the solution ofNdeI-digested plasmid pM8BW27 DNA, and the resulting reaction wasincubated at 37 ° C. for 2 hours. The EcoRI-NdeI-digested plasmidpM8BW27 DNA was electrophoresed on an agarose gel until the ˜560 byNdeI-EcoRI restriction fragment, which contains the portion of TPAcoding sequence that spans the site of deletion, was separated from theother digestion products. The ˜560 bp NdeI-EcoRI restriction fragmentwas isolated from the gel; about 0.5 μg of the desired fragment wasobtained and suspended in 20 μl of glass-distilled H₂O.

The second fragment needed to construct plasmid pBW28 is synthesized onestrand at a time on an automated DNA synthesizer. The two complementarystrands, which will hybridize to form a double-stranded DNA segment withXbaI and NdeI overlaps, are kinased and annealed in substantialaccordance with the procedure of Example 6A. The linker has thefollowing structure:

The third fragment needed to construct plasmid pBW28 was prepared byadding ˜20 μg of plasmid pTPA103 in 20 μl of TE buffer to 10 μl of 10×BamHI buffer and 60 μl of H₂O. About 10 μl (˜50 units) of restrictionenzyme BamHI were added to the solution of plasmid pTPA103 DNA, and theresulting reaction was incubated at 37° C. for 2 hours. TheBamHI-digested plasmid pTPA103 DNA was precipitated with ethanol,collected by centrifugation, and resuspended in 10 μl of 10× EcoRIbuffer and 80 μl of H₂O. About 10 μl (˜50 units) of restriction enzymeEcoRI were added to the solution of BamHI-digested plasmid pTPA103 DNA,and the resulting reaction was incubated at 37° C. for 2 hours. TheBamHI-EcoRI-digested plasmid pTPA103 DNA was loaded onto an agarose geland electrophoresed until the ˜689 bp EcoRI-BamHI restriction fragment,which comprises the coding sequence for the carboxy-terminus of TPA, wasseparated from the other digestion products. About 0.5 μg of the ˜689 bpfragment was isolated from the gel and then resuspended in 10 μl ofglass-distilled H₂O.

The final fragment necessary to construct plasmid pBW28 was isolatedfrom plasmid pL110, which is a plasmid disclosed and claimed in U.S.patent application Ser. No. 769,221, filed Aug. 26, 1985, attorneydocket number X-6638. A restriction site and function map of plasmidpL110 is presented in FIG. 14 of the accompanying drawings, and theconstruction of plasmid pL110 is disclosed in Example 10d, the followingsection of the present Example.

About 25 μg of plasmid pL110 in 25 μl of TE buffer were added to 10 μlof 10× XbaI buffer (0.5M NaCl; 60 mM Tris-HCl, pH=7.9; 60 mM MgCl₂; and1 mg/ml BSA) and 55 μl of H₂O. About 10 μl (˜50 units) of restrictionenzyme XbaI were added to the solution of plasmid pL110 DNA, and theresulting reaction was incubated at 37° C. for 2 hours. TheXbaI-digested plasmid pL110 DNA was precipitated with ethanol, collectedby centrifugation, and resuspended in 10 μl of 10× BamHI buffer and 89μl of H₂O. About 1 μl (˜5 units) of restriction enzyme BamHI was addedto the solution of XbaI-digested plasmid pL110 DNA, and the resultingreaction was incubated at 37° C. for 30 minutes to obtain a partialBamHI digest. The XbaI-partially-BamHI-digested plasmid-pL110 DNA wasloaded onto a agarose gel and electrophoresed until the ˜6.0 kbXbaI-BamHI fragment was clearly separated from the other digestionproducts. The ˜6.0 kb restriction fragment was isolated from the gel;about 0.5 μg of the ˜6.0 kb XbaI-BamHI restriction fragment was obtainedand suspended in about 40 μl of glass-distilled H₂O. This ˜6.0 kbXbaI-BamHI restriction fragment comprises all of plasmid pL110 exceptthe EK-BGH-encoding DNA.

To construct plasmid pBW28, the following fragments are mixed together:about 0.1 μg (˜8 μl) of the ˜6.0 kb BamHI-XbaI restriction fragment ofplasmid pL110; about 0.05 μg (˜2 μl) of the ˜560 bp NdeI-EcoRIrestriction fragment of plasmid pM8BW27; about 0.1 μg (˜2 μl) of the˜689 bp EcoRI-BamHI restriction fragment of plasmid pTPA103; and about0.02 μg (˜1 μl) of the ˜45 bp XbaI-NdeI synthetic linker. About 2 μl of10× ligase buffer and 1 μl (˜1 Weiss unit) of T4 DNA ligase are added tothe mixture of DNA, and the resulting ligation reaction is incubated at4° C. overnight for 2 hours. The ligated DNA constituted the desiredplasmid pBW28. A restriction site and function map of plasmid pBW28 ispresented in FIG. 14 of the accompanying drawings.

The ligated DNA was used to transform E. coli K12MM294 (NRRL B-15625)made competent in substantial accordance with the procedure of Example2, except that 50 mM CaCl₂ was used in the procedure. Due to thepresence of the lambda pL promoter and the gene encoding thetemperature-sensitive lambda pL repressor on plasmid pBW28, thetransformation procedure and culturing of transformants were variedsomewhat. The cells were not exposed to temperatures greater than 32° C.during transformation and subsequent culturing. The following section ofthis Example relates more fully the procedures for handling plasmidsthat encode the lambda pL promoter and its temperature-sensitiverepressor. The desired E. coli K12MM294/pBW28 transformants wereidentified by their tetracycline-resistant, ampicillin-sensitivephenotype and by restriction enzyme analysis of their plasmid DNA.

D. Construction of Plasmid pL110

Plasmid pL110 was constructed using plasmid pKC283 as starting material.Lyophils of E. coli K12 BE1201/pKC283 are obtained from the NRRL underthe accession number NRRL B-15830. The lyophils are decanted into tubescontaining 10 ml of L broth and incubated two hours at 32° C., at whichtime the cultures are made 50 μg/ml in ampicillin and then incubated at32° C. overnight. The E. coli. K12 BE1201/pKC283 cells were cultured at32° C., because plasmid pKC283 comprises the pL promoter and because E.coli K12 BE1201 cells comprise a temperature-sensitive cI repressor geneintegrated into the cellular DNA. When cells that comprise a wild-typelambda pL repressor gene or when cells that do not comprise a lambda pLpromoter are utilized in this plasmid isolation procedure, as describedin subsequent Examples herein, the temperature of incubation is 37° C.

A small portion of the overnight culture is placed on L-agar platescontaining 50 μg/ml ampicillin in a manner so as to obtain a singlecolony isolate of E. coli K12 BE1201/pK283. The single colony obtainedwas inoculated into 10 ml of L broth containing 50 μg/ml ampicillin andincubated overnight at 32° C. with vigorous shaking. The 10 ml overnightculture was inoculated into 500 ml of L broth and incubated at 32° C.with vigorous shaking until the culture reached stationary phase.Plasmid pKC283 DNA was then prepared from the cells in substantialaccordance with the procedure of Example 3. About 1 mg of plasmid pKC283was obtained and stored at 4° C. in TE buffer at a concentration ofabout 1 μg/ul. A restriction site and function map of plasmid pKC283 ispresented in FIG. 14 of the accompanying drawings.

About 10 μl (˜10 μg) of the plasmid pKC283 DNA were mixed with 20 μl 10×medium-salt restriction buffer (500 mM NaCl; 100 mM Tris-HCl, pH=7.5;100 mM MgCl₂; and 10 mM DTT), 20 μl mg/ml BSA, 5 μl restriction enzymePvuII (˜25 units), and 145 μl of water, and the resulting reaction wasincubated at 37° C. for 2 hours. Restriction enzyme reactions describedherein were routinely terminated by phenol and then chloroformextractions, which were followed by precipitation of the DNA, an ethanolwash, and resuspension of the DNA in TE buffer. After terminating thePvuII digestion as described above, the PvuII-digested plasmid pKC283DNA was precipitated and then resuspended in 5 μl of TE buffer.

About 600 picomoles (pM) of XhoI linkers (5′-CCTCGAGG-3′) were kinasedin a mixture containing 10 μl of 5× Kinase Buffer (300 mM Tris-HCl,pH=7.8; 50 mM MgCl₂; and 25 mM DTT), 5 μl of 5 mM ATP, 24 μl of H₂O, 0.5μl of T4 polynucleotide kinase (about 2.5 units as defined by P-LBiochemicals), 5 μl of 1 mg/ml BSA, and 5 μl of 10 mM spermidine byincubating the mixture at 37° C. for 30 minutes. About 12.5 μl of thekinased XhoI linkers were added to the 5 μl of PvuII-digested plasmidpKC283 DNA, and then, 2.5 μl of 10× ligase buffer, 2.5 μl (about 2.5units as defined by P-L Biochemicals) of T4 DNA ligase, 2.5 μl of 10 mMspermidine, and 12.5 μl of water were added to the DNA. The resultingligation reaction was incubated at 4° C. overnight. After the ligationreaction, the reaction mixture was adjusted to have the composition ofhigh-salt buffer (0.1M NaCl; 0.05M Tris-HCl, pH 7.5; 10.0 mM MgCl₂; and1 mM DTT). About 10 μl (100 units) of restriction enzyme XhoI were addedto the mixture, and the resulting reaction was incubated at 37° C. for 2hours.

The reaction was terminated, and the XhoI-digested DNA was precipitated,resuspended, and ligated as described above, except that no XhoI linkerswere added to the ligation mixture. The ligated DNA constituted thedesired plasmid pKC283PX. A restriction site and function map of plasmidpKC283PX is presented in FIG. 14 of the accompanying drawings.

E. coli K12MO(λ⁺), available from the NRRL under the accession numberNRRL B-15993, comprises the wild-type lambda pL cI repressor gene, sothat transcription from the lambda pL promoter does not occur in E. coliK12MO(λ⁺) cells. Single colonies of E. coli K12MO(λ⁺) are isolated, anda 10 ml overnight culture of the cells is prepared; no ampicillin isused in the growth media. Fifty μl of the overnight culture were used toinoculate 5 ml of L broth, which also contained 10 mM MgSO₄ and 10 mMMgCl₂. The culture was incubated at 37° C. overnight with vigorousshaking. The following morning, the culture was diluted to 200 ml with Lbroth containing 10 mM MgSO₄ and 10 mM MgCl₂. The diluted culture wasincubated at 37° C. with vigorous shaking until the O.D.₅₉₀ was about0.5, which indicated a cell density of about 1×10⁸ cells/ml. The culturewas cooled for ten minutes in an ice-water bath, and the cells were thencollected by centrifugation at 4000×g for 10 minutes at 4° C. The cellpellet was resuspended in 100 ml of cold 10 mM NaCl and then immediatelyre-pelleted by centrifugation. The cell pellet was resuspended in 100 mlof 30 mM CaCl₂ and incubated on ice for 20 minutes.

The cells were again collected by centrifugation and resuspended in 10ml of 30 mM CaCl₂. A one-half ml aliquot of the cells was added to theligated DNA prepared above; the DNA had been made 30 mM in CaCl₂. Thecell-DNA mixture was incubated on ice for one hour, heat-shocked at 42°C. for 90 seconds, and then chilled on ice for about two minutes. Thecell-DNA mixture was diluted into 10 ml of LB media in 125 ml flasks andincubated at 37° C. for one hour. One hundred μl aliquots were plated onL-agar plates containing ampicillin and incubated at 37° C. untilcolonies appeared.

The colonies were individually cultured, and the plasmid DNA of theindividual colonies was examined by restriction enzyme analysis and gelelectrophoresis. Plasmid DNA isolation was performed on a smaller scalein accordance with the procedure of Example 3, but the CsCl gradientstep was omitted until the desired E. coli K12MO(λ⁺)/pKC283PXtransformants were identified. A restriction site and function map ofplasmid pKC283PX is presented in FIG. 14 of the accompanying drawings.

Ten μg of plasmid pKC283PX DNA were dissolved in 20 μl of 10× high-saltbuffer, 20 μl 1 mg/ml BSA, 5 μl (˜50 units) of restriction enzyme BglII,5 μl (˜50 units) of restriction enzyme XhoI, and 150 μl of water, andthe resulting reaction was incubated at 37° C. for two hours. Thereaction was stopped; the BglII-XhoI digested DNA was precipitated, andthe DNA was resuspended in 5 μl of TE buffer.

A DNA linker with single-stranded DNA ends characteristic of BglII andXhoI restriction enzyme cleavage was synthesized using an automated DNAsynthesizer and kinased as described in Example 6A. The DNA linker hadthe following structure:

The linker and BglII-XhoI-digested plasmid pKC283PX were ligated insubstantial accordance with the ligation procedure described above. Theligated DNA constituted the desired plasmid pKC283-L. A restriction siteand function map of plasmid pKC283-L is presented in FIG. 14 of theaccompanying drawings. The plasmid pKC183-L DNA was used to transform E.coli K12MO(λ⁺), and the resulting E. coli K12MO(λ⁺)/pKC283-Ltransformants were identified by their ampicillin-resistant phenotypeand by restriction enzyme analysis of their plasmid DNA.

About 10 μg of plasmid pKC183-L DNA were dissolved in 20 μl 10×high-salt buffer, 20 μl 1 mg/ml BSA, 5 μl (˜50 units) restriction enzymeXhoI, and 155 μl of H₂O, and the resulting reaction was incubated at 37°C. for two hours. The XhoI-digested plasmid pKC283-L DNA was thenprecipitated and resuspended in 2 μl 10× nick-translation buffer (0.5MTris-HCl, pH=7.2; 0.1M MgSO₄; and 1 mM DTT), 1 μl of a solution 2 mM ineach of the deoxynucleotide trisphosphates, 15 μl of H₂O, 1 μl (˜6 unitsas defined by P-L Biochemicals) of Klenow, and 1 μl of 1 mg/ml BSA. Theresulting reaction was incubated at 25° C. for 30 minutes; the reactionwas stopped by incubating the solution at 70° C. for five minutes.

BamHI linkers (5¹-CGGGATCCCG-3′) were kinased and ligated to theXhoI-digested, Klenow-treated plasmid pKC183-L DNA in substantialaccordance with the linker ligation procedures described above. Afterthe ligation reaction, the DNA was digested with about 100 units ofBamHI for about 2 hours at 37° C. in high-salt buffer. After the BamHIdigestion, the DNA was prepared for ligation, and the ˜5.9 kb BamHIrestriction fragment was circularized by ligation and transformed intoE. coli K12MO(λ⁺) in substantial accordance with the proceduresdescribed above. The E. coli K12MO(λ⁺)/pKC283-LB transformants wereidentified, and then, plasmid pKC283-LB DNA was prepared from thetransformants in substantial accordance with the procedure of Example 3.A restriction site and function map of plasmid pKC283-LB is presented inFIG. 14 of the accompanying drawings.

About 10 μg of plasmid pKC283PX were digested with restriction enzymeSalI in high-salt buffer, treated with Klenow, and ligated to EcoRIlinkers (5¹-GAGGAATTCCTC-3′) in substantial accordance with theprocedures described above. After digestion with restriction enzymeEcoRI, which results in the excision of ˜2.1 kb of DNA, the ˜4.0 kbEcoRI restriction fragment was circularized by ligation to yield plasmidpKC283PRS. The ligated DNA was used to transform E. coli K12MO(λ⁺), andafter the E. coli K12MO(λ⁺)/pKC283PRS transformants were identified,plasmid pKC283PRS DNA was prepared from the transformants, insubstantial accordance with the procedure of Example 3. A restrictionsite and function map of plasmid pKC283PRS is presented in FIG. 14 ofthe accompanying drawings.

About 10 μg of plasmid pKC283PRS were digested in 200 μl of high-saltbuffer with about 50 units each of restriction enzymes PstI and SPhI.After incubating the reaction at 37° C. for about 2 hours, the reactionmixture was electrophoresed on a 0.6% low-gelling-temperature agarose(FMC Corporation, Marine Colloids Division, Rockland, Me. 04841) gel for2-3 hours at ˜130 V and ˜75 mA in Tris-Acetate buffer.

The gel was stained in a dilute solution of ethidium bromide, and theband of DNA constituting the ˜0.85 kb PstI-SphI restriction fragment,which was visualized with long-wave UV light, was cut from the gel in asmall segment. The volume of the segment was determined by weight anddensity of the segment, and an equal volume of 10 mM Tris-HCl, pH 7.6,was added to the tube containing the segment. The segment was thenmelted by incubation at 72° C. About 1 ug of the ˜0.85 kb PstI-SphIrestriction fragment of plasmid pKC283PRS was obtained in a volume ofabout 100 μl. In an analogous manner, plasmid pKC183-LB was digestedwith restriction enzymes PstI and SphI, and the resulting ˜3.0 kbrestriction fragment was isolated by agarose gel electrophoresis andprepared for ligation.

The ˜0.85 kb PstI-SphI restriction fragment of plasmid pKC183PRS wasligated to the ˜3.0 kb PstI-SPhI restriction fragment of plasmidpKC283-LB. The ligated DNA constituted the desired plasmid pL32. Arestriction site and function map of plasmid pL32 is presented in FIG.14 of the accompanying drawings. Plasmid pL32 was transformed into E.coli K12MO(λ⁺) cells; plasmid pL32 DNA was prepared from the E. coliK12MO(λ⁺)/pL32 transformants in substantial accordance with theprocedure of Example 3. Analysis of the plasmid pL32 DNA demonstratedthat more than one EcoRI linker attached to the Klenow-treated, SalIends of plasmid pKC283PX. The presence of more than one EcoRI linkerdoes not affect the utility of plasmid pL32 or derivatives of plasmidpL32 and can be detected by the presence of an XhoI restriction site,which is generated whenever two of the EcoRI linkers are ligatedtogether.

Plasmid pCC101 is disclosed in Example 3 of U.S. patent application Ser.No. 586,581, filed Mar. 6, 1984, attorney docket number X-5872A,incorporated herein by reference. A restriction site and function map ofplasmid pCC101 is presented in FIG. 14 of the accompanying drawings. Toisolate the EK-BGH-encoding DNA, about 10 μg of plasmid pCC101 weredigested in 200 μl of high-salt buffer containing about 50 units each ofrestriction enzymes XbaI and BamHI. The digestion products wereseparated by agarose gel electrophoresis, and the ˜0.6 kb XbaI-BamHIrestriction fragment which encodes EK-BGH was isolated from the gel andprepared for ligation.

Plasmid pL32 was also digested with restriction enzymes XbaI and BamHI,and the ˜3.9 kb restriction fragment was isolated and prepared forligation. The ˜3.9 kb XbaI-BamHI restriction fragment of plasmid pL32was ligated to the ˜0.6 kb XbaI-BamHI restriction fragment of plasmidpCC101 to yield plasmid pL47. A restriction site and function map ofplasmid pL47 is presented in FIG. 14 of the accompanying drawings.Plasmid pL47 was transformed into E. coli K12MO(λ⁺), and the E. coliK12MO(λ⁺)/pL47 transformants were identified. Plasmid pL47 DNA wasprepared from the transformants in substantial accordance with theprocedures of Example 3.

Plasmid pPR12 comprises the temperature-sensitive pL repressor genecI857 and the plasmid pBR322 tetracycline resistance-conferring gene.Plasmid pPR12 is disclosed and claimed in U.S. Pat. No. 4,436,815,issued Mar. 13, 1984. A restriction site and function map of plasmidpPR12 is presented in FIG. 14 of the accompanying drawings.

About 10 μg of plasmid pPR12 were digested with about 50 units ofrestriction enzyme EcoRI in 200 μl of high-salt buffer at 37° C. for twohours. The EcoRI-digested plasmid pPR12 DNA was precipitated and thentreated with Klenow in substantial accordance with the proceduredescribed above. After the Klenow reaction, the EcoRI-digested,Klenow-treated plasmid pPR12 DNA was recircularized by ligation, and theligated DNA, which constituted the desired plasmid pPR12ΔR1, used totransform E. coli K12 RV308 (NRRL B-15624); transformants were selectedbased on tetracycline (10 ug/ml) resistance. After the E. coli K12RV308/pPR12ΔR1 transformants were identified, plasmid pPR12ΔR1 DNA wasprepared from the transformants in substantial accordance with theprocedure of Example 3.

About 10 μg of plasmid pPR12ΔR1 were digested with about 50 units ofreaction enzyme AvaI in 200 μl of medium-salt buffer at 37° C. for 2hours. The AvaI-digested plasmid pPR12ΔR1 DNA was precipitated and thentreated with Klenow. After the Klenow reaction, the AvaI-digested,Klenow-treated plasmid pPR12ΔR1 DNA was ligated to EcoR1 linkers(5′-GAGGAATTCCTC-3′), precipitated, resuspended in about 200 μl ofhigh-salt buffer containing about 50 units of restriction enzyme EcoRI,and incubated at 37° C. for about 2 hours. After the EcoR1 digestion,the reaction mixture was loaded onto a low-melting agarose gel, and the˜5.1 kb EcoR1 restriction fragment was purified from the gel andrecircularized by ligation to yield the desired plasmid pPR12AR1. Theplasmid pPR12AR1 DNA was transformed into E. coli K12 RV308; selectionof transformants was based on tetracycline resistance. Plasmid pPR12AR1DNA prepared from the transformants in substantial accordance with theprocedure of Example 3. A restriction site and function map of plasmidpPR12AR1 is presented in FIG. 14 of the accompanying drawings.

About 10 μg of plasmid pPR12R1 DNA were suspended in about 200 ml ofhigh-salt buffer containing about 50 units each of restriction enzymesPstI and EcoRI, and the digestion reaction mixture was incubated at 37°C. for about 2 hours. The reaction mixture was then loaded onto anagarose gel, and the ˜2.9 kb PstI-EcoR1 restriction fragment of plasmidpPR12AR1 was isolated and prepared for ligation.

About 10 ug of plasmid pL47 were digested with restriction enzymes PstIand BamHI in 200 ul of high-salt buffer at 37° C. for two hours. ThePstI-BamHI-digested DNA was loaded onto an agarose gel, and the ˜2.7 kbPstI-BamHI restriction fragment that comprised the origin of replicationand a portion of the ampicillin resistance-conferring gene was isolatedand prepared for ligation. In a separate reaction, about 10 ug ofplasmid pL47 DNA were digested with restriction enzymes EcoRI and BamHIin 200 ul of high-salt buffer at 37° C. for two hours, and the ˜1.03 kbEcoRI-BamHI restriction fragment that comprised the lambda pLtranscription activating sequence, the E. coli lpp translationactivating sequence, and the EK-BGH-encoding DNA was isolated andprepared for ligation.

The ˜2.7 kb PstI-BamHI and ˜1.03 kb EcoRI-BamHI restriction fragments ofplasmid pL47 were ligated to the ˜2.9 kb PstI-EcoRI restriction fragmentof plasmid pPR12AR1 to construct plasmid pL110, and the ligated DNA wasused to transform E. coli K12 RV308. Tetracycline resistance was used asthe basis for selecting transformants.

Two PstI restriction enzyme recognition sites are present in the EK-BGHcoding region that are not depicted in the restriction site and functionmaps presented in the accompanying drawings. A restriction site andfunction map of plasmid pL110 is presented in FIG. 14 of theaccompanying drawings.

E. Final Construction of Plasmid pBW32

Approximately 10 μg of plasmid pSV2-β-globin DNA (NRRL B-15928) weredissolved in 10 μl 10× HindIII reaction buffer, 5 μl (˜50 units)restriction enzyme HindIII, and 85 μl H₂O, and the reaction was placedat 37° C. for 2 hours. The reaction mixture was then made 0.15M in LiCl,and after the addition of 2.5 volumes of ethanol and incubation in a dryice-ethanol bath, the DNA was pelleted by centrifugation.

The DNA pellet was dissolved in 10 μl 10× BglII buffer, 5 μl (˜50 units)restriction enzyme BglII, and 85 μl H₂O, and the reaction was placed at37° C. for two hours. After the BglII digestion, the reaction mixturewas loaded onto a 0.85% agarose gel, and the fragments were separated byelectrophoresis. The gel was visualized using ethidium bromide andultraviolet light, and the band containing the desired ˜4.2 kbHindIII-BglII fragment was excised from the gel as previously described.The pellet was resuspended in 10 μl of H₂O and constituted ˜5 μg of thedesired ˜4.2 kb HindIII-BglII restriction fragment of plasmidpSV2-β-globin. The ˜2.0 kb HindIII-BamHI restriction fragment of plasmidpTPA103 that encodes TPA was isolated from plasmid pTPA103 insubstantial accordance with the foregoing teaching. About 5 μg of the˜2.0 kb HindIII-BamHI restriction fragment of plasmid pTPA103 wereobtained, suspended in 10 μl of H₂O, and stored at −20° C.

Two μl of the ˜4.2 kb BglII-HindIII restriction fragment of plasmidpSV2-β-globin and 4 μl of the ˜2.0 kb HindIII-BamHl fragment of plasmidpTPA103 were mixed together and then incubated with 2 μl of 10× ligasebuffer, 11 μl of H₂O, and 1 μl of T4 DNA ligase (˜500 units) at 4° C.overnight. The ligated DNA constituted the desired plasmid pTPA301; arestriction site and function map of the plasmid is presented in FIG. 14of the accompanying drawings. The ligated DNA was used to transform E.coli K12 RR1 cells (NRRL B-15210) made competent for transformation insubstantial accordance with the teaching of Example 3. Plasmid DNA wasobtained from the E. coli K12 RR1/pTPA301 transformants in substantialaccordance with the procedure of Example 3.

Plasmid pSV2-dhfr comprises a dihydrofalate reductase (dhfr) gene usefulfor selection of transformed eukaryotic cells and amplification of DNAcovalently linked to the dhfr gene. Ten μg of plasmid pSV2-dhfr(isolated from E. coli K12 HB101/pSV2-dhfr, ATCC 37146) were mixed with10 μl 10× PvuII buffer, 2 μl (˜20 units) PvuII restriction enzyme, and88 μl of H₂O, and the resulting reaction was incubated at 37° C. for twohours. The reaction was terminated by phenol and chloroform extractions,and then, the PvuII-digested plasmid pSV2-dhfr DNA was precipitated andcollected by centrifugation.

BamHI linkers (5′-CGGATCCCG-3′) were kinased and prepared for ligationby the following procedure. To 1 μg of linker in 5 μl H₂O was added: 10μl 5× Kinase salts (300 mM Tris-HCl, pH=7.8; 50 mM MgCl₂; and 25 mMDTT), 5 μl of 5 mM ATP, 5 μl of BSA (1 mg/ml), 5 μl of 10 mM spermidine,19 μl of H₂O, and 1 μl of polynucleotide Kinase (10 units/μl). Thisreaction was then incubated at 37° C. for 60 minutes and stored at −20°C. Five μl (˜5 μg) of the PvuII-digested plasmid pSV2-dhfr and 12 μl(μ0.25 μg ) of the kinased BamHI linkers were mixed and incubated with11 μl of H₂O, 2 μl 10× ligase buffer, and 1 μl (˜1000 units) of T4 DNAligase at 16° C. overnight.

Ten μl of 10× BamHI reaction buffer, 10 μl (˜50 units) of BamHIrestriction enzyme, and 48 μl of H₂O were added to the ligation reactionmixture, which was then incubated at 37° C. for 3 hours. The reactionwas loaded onto a 1% agarose gel, and the desired ˜1.9 kb fragment,which comprises the dhfr gene, was isolated from the gel. All linkeradditions performed in these examples were routinely purified on anagarose gel to reduce the likelihood of multiple linker sequences in thefinal vector. The ˜3 μg of fragment obtained were suspended in 10 μl ofTE buffer.

Next, approximately 15 μl (˜1 μg) of plasmid pTPA301 were digested withBamHI restriction enzyme as taught above. Because there is a uniqueBamHI site in plasmid pTPA301, this BamHI digestion generates linearplasmid pTPA301 DNA. The BamHI-digested plasmid pTPA301 was precipitatedwith ethanol and resuspended in 94 μl of H₂O and phosphatased using 1 μlof Calf-Intestinal Alkaline phosphatase (Collaborative Research, Inc.,128 Spring Street, Lexington, Mass. 02173), and 5 μl of 1M Tris-HCl,pH=9.0, at 65° C. for 45 min. The DNA was extracted withphenol:chloroform, then extracted with chloroform:isoamyl alcohol,ethanol precipitated, and resuspended in 20 μl H₂O. Ten μl (˜0.25 μg) ofphosphatased plasmid pTPA301 were added to 5 μof the BamHI,dhfr-gene-containing restriction fragment (˜1.5 μg), 3 μl of 10× ligasebuffer, 3 μl (˜1500 units) of T4 DNA ligase, and 9 μl H₂O. This ligationreaction was incubated at 15° C. overnight; the ligated DNA constitutedthe desired plasmid pTPA303 DNA.

Plasmid pTPA303 was used to transform E. coli K12 RR1 (NRRL B-15210),and the resulting E. coli K12 RR1/pTPA303 transformants were identifiedby their ampicillin-resistant phenotype and by restriction enzymeanalysis of their plasmid DNA. Plasmid pTPA303 was isolated from thetransformants in substantial accordance with the procedure of Example 3.

To isolate the ˜2.7 kb EcoRI-BglII restriction fragment that encodes thepBR322 replicon and β-lactamase gene from plasmid pTPA301, about 10 μgof plasmid pTPA301 are digested to completion in 400 μl total reactionvolume with 20 units BglII restriction enzyme in 1× BglII buffer at 37°C. after the BglII digestion, the Tris-HCl concentration is adjusted to110 mM, and 20 units of EcoRI restriction enzyme are added to theBglII-digested DNA. The EcoRI-BglII-digested DNA is loaded onto anagarose gel and electrophoresed until the ˜2.7 kb EcoRI-BglIIrestriction fragment is separated from the other digestion products, andthen, the ˜2.7 kb fragment is isolated and prepared for ligation.

To isolate a restriction fragment that comprises the dhfr gene, plasmidpTPA303 was double-digested with HindIII and EcoRI restriction enzymes,and the ˜2340 bp EcoRI-HindIII restriction fragment that comprises thedhfr gene was isolated and recovered.

To isolate the ˜2 kb HindIII-SstI restriction fragment of plasmidpTPA303 that comprises the coding region for the carboxy-terminus of TPAand the SV40 promoter, plasmid pTPA303 was double digested with HindIIIand SstI restriction enzymes in 1× HindIII buffer. The ˜1.7 kb fragmentwas isolated from the gel and prepared for ligation.

To isolate the ˜680 bp XhoII (compatible for ligation with the BglIIoverlap)-SstI restriction fragment of plasmid pBW28 that comprises thecoding region for the amino terminus of modified TPA, about 10 μg ofplasmid pBW28 were digested with XhoII enzyme to completion in 1× XhoIIbuffer (0.1M Tris-HCl, pH=8.0; 0.1M MgCl₂; 0.1% Triton X-100; and 1mg/ml BSA). The XhoII-digested DNA was recovered by ethanolprecipitation and subsequently digested to completion with SstI enzyme.The XhoII-SstI-digested DNA was loaded onto an acrylamide gel, and thedesired fragment was isolated from the gel and prepared for ligation.

About 0.1 μg of each of the above fragments: the ˜2.7 kb EcoRI-BglIIrestriction fragment of plasmid pTPA301; the ˜2.34 kb EcoRI-HindIIIrestriction fragment of plasmid pTPA303; the ˜1.7 kb SstI-HindIIIrestriction fragment of plasmid pTPA303; and the ˜0.68 kb SstI-XhoIIrestriction fragment of plasmid pBW28 were ligated together to formplasmid pBW32. The ligation mix was used to transform E. coli K12MM294as taught in Example 2, except that 50 mM CaCl₂ was used in theprocedure. Transformants were identified by their ampicillin-resistantphenotype and by restrictified analysis of their plasmid DNA. PlasmidpBW32 DNA was obtained from the E. coli K12MM294/pBW32 transformants insubstantial accordance with the procedure of Example 3. A restrictionsite and function map of plasmid pBW32 is presented in FIG. 14 of theaccompanying drawings.

EXAMPLE 11 Construction of Plasmids pLPChd1, pLPChd2, pLPCdhfr1, andpLPCdhfr2

A. Construction of Plasmids pLPChd1 and pLPChd2

About 20 μg of plasmid pBW32 in 20 μl of TE buffer were added to 10 μlof 10× BamHI buffer and 60 μl of H₂O. About 10 μl (˜50 units) ofrestriction enzyme BamHI were added to the solution of plasmid pBW32DNA, and the resulting reaction was incubated at 37° C. for two hours.The BamHI-digested plasmid pBW32 DNA was precipitated with ethanol,collected by centrifugation, and resuspended in 5 μl of 10× Klenowbuffer, 45 μl of H₂O, and 2 μl (˜100 units) of Klenow enzyme. Thereaction was incubated at 16° C. for 30 minutes; then, the reactionmixture was loaded onto an agarose gel and electrophoresed until thedigestion products were clearly separated. The ˜1.9 kb Klenow-treated,BamHI restriction fragment of plasmid pBW32 that comprises the dhfr genewas isolated from the gel and prepared for ligation in substantialaccordance with the procedure of Example 4A. About 4 μg of the desiredfragment were obtained and suspended in 5 μl of TE buffer.

About 200 μg of plasmid pLPChyg1 in 100 μl of TE buffer were added to 15μl of 10× EcoRI buffer and 30 μl of H₂O. About 5 μl (˜50 units) ofrestriction enzyme EcoRI were added to the solution of plasmid pLPChyg1DNA, and the resulting reaction was incubated at 37° C. for about 10minutes. The short reaction time was calculated to produce a partialEcoRI digestion. Plasmid pLPChyg1 has two EcoRI restriction sites, oneof which is within the coding sequence of the hygromycinresistance-conferring (HmR) gene, and it was desired to insert thedhfr-gene-containing restriction fragment into the EcoRI site of plasmidpLPChyg1 that is not in the HmR gene. The partially-EcoRI-digestedplasmid pLPChyg1 DNA was loaded onto an agarose gel and electrophoreseduntil the singly-cut plasmid pLPChyg1 DNA was separated from uncutplasmid DNA and the other digestion products. The singly-cut DNA wasisolated from the gel and prepared for ligation in substantialaccordance with the procedure of Example 4A. About 2 μg of thesingly-EcoRI-cut plasmid pLPChyg1 were obtained and suspended in 25 μlof TE buffer. To this sample, about 5 μl (˜25 units) of Klenow enzyme, 5μl of 10× Klenow buffer, and 40 μl of H₂O were added, and the resultingreaction was incubated at 16° C. for 60 minutes. The Klenow-treated,partially-EcoRI-digested DNA was then extracted twice with phenol andthen once with chloroform, precipitated with ethanol, and resuspended in25 μl of TE buffer.

About 5 μl of the ˜1.9 Klenow-treated BamHI restriction fragment ofplasmid pBW32 and about 5 μl of the singly-EcoRI-cut plasmid pLPChyg1DNA were mixed together, and 1 μl of 10× ligase buffer, 5 μl of H₂O, 1μl (˜500 units) of T4 DNA ligase, and 1 μl (˜2 units) of T4 RNA ligasewere added to the mixture of DNA, and the resulting reaction wasincubated at 16° C. overnight. The ligated DNA constituted the desiredplasmids pLPChd1 and pLPChd2, which differ only with respect to theorientation of the ˜1.9 kb fragment that comprises the dhfr gene.

The ligated DNA was used to transform E. coli K12 HB101 cells madecompetent for transformation in substantial accordance with theprocedure of Example 2. The transformed cells were plated onto L agarcontaining 100 μg /ml ampicillin, and the ampicillin-resistanttransformants were analyzed by restriction enzyme analysis of theirplasmid DNA to identify the E. coli K12 HB101/pLPChd1 and E. coli K12HB101/pLPChd2 transformants. A restriction site and function map ofplasmid pLPChd1 is presented in FIG. 15 of the accompanying drawings.Plasmid pLPChd1 and plasmid pLPChd 2 DNA were isolated from theappropriate transformants in substantial accordance with the procedureof Example 3.

Plasmids pLPChd3 and pLPChd4 are similar in structure to plasmidspLPChd1 and pLPChd2. Plasmids pLPChd3 and pLPChd4 are constructed insubstantial accordance with the procedure used to construct plasmidspLPChd1 and pLPChd2, except plasmid pLPChyg2 is used as startingmaterial in the procedure rather than plasmid pLPChyg1.

B. Construction of Plasmids pLPCdhfr1 and pLPCdhfr2

About 100 μg of plasmid pBW32 in 100 μl of TE buffer were added to 15 μlof 10× BamHI buffer and 25 μl of H₂O. About 10 μl (˜25 units) ofrestriction enzyme BamHI were added to the solution of plasmid pBW32DNA, and the resulting reaction was incubated at 37° C. for 2 hours. TheBamHI-digested plasmid pBW32 DNA was treated with Klenow in substantialaccordance with the procedure in Example 11A. The blunt-ended fragmentwas precipitated with ethanol, resuspended in 10 μl of TE buffer, loadedonto an agarose gel, and electrophoresed until the ˜1.9 kb BamHIrestriction fragment that comprises the dihydrofolate reductase gene wasseparated from the other digestion products. The ˜1.9 kb restrictionfragment was then isolated from the gel and prepared for ligation insubstantial accordance with the procedure of Example 4A; about 10 μg ofthe desired fragment were obtained and suspended in 50 μl of TE buffer.

About 5 μl of NdeI-StuI-digested plasmid pLPC DNA, as prepared inExample 9, were added to 5 μl of the Klenow-treated, ˜1.9 kb BamHIrestriction fragment of plasmid pBW32, 1.5 μl of 10× ligase buffer, 1 μl(˜1000 units) of T4 DNA ligase, 1 μl (˜2 units) of T4 RNA ligase, and1.5 μl of H₂O. The resulting ligation reaction was incubated at 16° C.overnight; the ligated DNA constituted the desired plasmids pLPCdhfr1and pLPCdhfr2, which differ only with respect to the orientation of the˜1.9 kb fragment that contains the dhfr gene. The ligated DNA was usedto transform E. coli K12 HB101 in substantial accordance with theprocedure of Example 2. The transformed cells were plated onto L agarcontaining ampicillin, and the ampicillin-resistant E. coli K12HB101/pLPCdhfr1 and E. coli K12 HB101/pLPCdhfr2 transformants wereidentified by restriction enzyme analysis of their plasmid DNA.

EXAMPLE 12 Construction of Plasmid phd

To construct plasmid phd, it was necessary to prepare the plasmidpLPChd1 DNA, used as starting material in the construction of plasmidphd, from E. coli host cells that lack an adenine methylase, such asthat encoded by the dam gene, the product of which methylates theadenine residue in the sequence 5′-GATC-3′. E. coli K12 GM48 (NRRLB-15725) lacks a functional dam methylase and so is a suitable host touse for the purpose of preparing plasmid pLPChd1 DNA for use as startingmaterial in the construction of plasmid phd.

E. coli K12 GM48 cells were cultured and made competent fortransformation, and plasmid pLPChyg1 was used to transform the E. coliK12 GM48 cells in substantial accordance with the procedure of Example2. The transformed cells were plated on L agar containing ampicillin,and once the ampicillin-resistant, E. coli K12 GM48/pLPChd1transformants had formed colonies, one such colony was used to prepareplasmid pLPChd1 DNA in substantial accordance with the procedure ofExample 3. About 1 mg of plasmid pLPChd1 DNA was obtained and suspendedin about 1 ml of TE buffer.

About 2 μl of plasmid pLPChd1 DNA in 2 μl of TE buffer were added to 2μl of 10× BclI buffer (750 mM KCl; 60 mM Tris-HCl, pH=7.4; 100 mM MgCl₂;10 mM DTT and 1 mg/ml BSA) and 14 μl of H₂O. About 2 μl (˜10 units) ofrestriction enzyme BclI were added to the solution of plasmid pLPChd1DNA, and the resulting reaction was incubated at 50° C. for two hours.The reaction was stopped by extracting the mixture once with phenol andtwice with chloroform.

About 1 μl of the BclI-digested plasmid pLPChd1 DNA was added to 1 μl of10× ligase buffer, 8 μl of H₂O and 1 μl (˜500 units) of T4 DNA ligase.The ligation reaction was incubated at 16° C. overnight, and the ligatedDNA constituted the desired plasmid phd. Plasmid phd results from thedeletion of the extra BclI linkers that attached during the constructionof plasmid pLPcat and the two adjacent BclI restriction fragments of atotal size of about 1.45 kb from plasmid pLPChd1. A restriction site andfunction map of plasmid phd is presented in FIG. 16 of the accompanyingdrawings. Plasmid phd facilitates the expression of any DNA sequencefrom the BK virus enhancer-adenovirus late promoter of the presentinvention, because the DNA to be expressed can be readily inserted inthe correct position for expression at the single BclI site on plasmidphd.

The ligated DNA was used to transform E. coli K12 GM48 in substantialaccordance with the procedure of Example 2. The transformed cells wereplated on L agar containing ampicillin, and the ampicillin-resistant E.coli K12 GM48/phd transformants were identified by restriction enzymeanalysis of their plasmid DNA.

Plasmids analogous to plasmid phd can be constructed in substantialaccordance with the foregoing procedure for constructing plasmid phdusing any of plasmids pLPChd2, pLPChd3, or pLPChd4 as starting materialrather than plasmid pLPChd1. These analagous plasmids differ fromplasmid phd only with respect to the orientation of the hygromycinresistance-conferring and/or dhfr genes.

EXAMPLE 13 Construction of Plasmid pLPCE1A

To isolate the E1A gene of adenovirus 2 DNA, about 20 μg of adenovirus 2DNA (from BRL) were dissolved in 10 μl of 10× BalI buffer (100 mMTris-HCl, pH=7.6; 120 mM MgCl₂; 100 mM 2-mercaptoethanol; and 1 mg/mlBSA) and 80 μl of H₂O. About 10 μl (about 20 units) of restrictionenzyme BalI were added to the solution of adenovirus 2 DNA, and theresulting reaction was incubated at 37° C. for two hours. TheBalI-digested DNA was loaded onto an agarose gel and electrophoreseduntil the ˜1.8 kb restriction fragment that comprises the E1A gene wasseparated from the other digestion products. The ˜1.8 kb fragment wasisolated from the gel and prepared for ligation in substantialaccordance with the procedure of Example 4A. About 3 μg of the desiredfragment was obtained and suspended in 20 μl of TE buffer.

About 5 μl of plasmid pLPC in 5 μl of TE buffer were added to 2 μl of10× StuI buffer and 11 μl of H₂O. About 2 μl (˜10 units) of restrictionenzyme StuI were added to the solution of plasmid pLPC, and theresulting reaction was incubated at 37° C. for 2 hours. TheStuI-digested plasmid pLPC DNA was precipitated with ethanol andresuspended in 2 μl of 10× NdeI buffer and 16 μl of H₂O. About 2 μl (˜10units) of restriction enzyme NdeI were added to the solution ofStuI-digested plasmid pLPC DNA, and the resulting reaction was incubatedat 37° C. for 2 hours.

The NdeI-StuI-digested plasmid PLPC DNA was precipitated with ethanoland resuspended in 5 μl of 10× Klenow buffer and 42 μl of H₂O. About 3μl (˜6 units) of Klenow enzyme were added to the solution of DNA, andthe resulting reaction was incubated at 37° C. for 30 minutes. Thereaction mixture was then loaded onto an agarose gel and electrophoreseduntil the ˜5.82 kb, Klenow-treated, NdeI-StuI restriction fragment wasclearly separated from the other reaction products. The fragment wasisolated from the gel and prepared for ligation in substantialaccordance with the procedure of Example 4A. About 2 μg of the ˜5.82 kb,Klenow-treated, NdeI-StuI restriction fragment of plasmid PLPC wereobtained and suspended in 25 μl of TE buffer.

About 9 μl of the ˜1.8 kb BalI restriction fragment of adenovirus 2 thatencodes the E1A gene and 3 μl of the ˜5.82 kb, Klenow-treated, NdeI-StuIrestriction fragment of plasmid pLPC were added to 2 μl of 1033 ligasebuffer and 4 μl of H₂O. About 1 μl (˜500 units) of T4 DNA ligase and 1μl (˜2 units) of T4 RNA ligase were added to the solution of DNA, andthe resulting reaction was incubated at 16° C. overnight.

The ligated DNA constituted the desired plasmids pLPCE1A and pLPCE1A1,which differ with respect to the orientation of the E1A gene andpossibly differ with respect to the expression-enhancing effect the BKenhancer has on the E1A gene on the plasmid. Because the E1A promoter islocated closer to the BK enhancer on plasmid pLPCE1A than plasmidpLPCE1A1, E1A expression may be higher when plasmid pLPCE1A is used asopposed to plasmid pLPCE1A1. A restriction site and function map ofplasmid pLPCE1A is presented in FIG. 17. of the accompanying drawings.

The ligated DNA was used to transform E. coli K12 HB101 in substantialaccordance with the procedure of Example 2. The transformed cells wereplated on L agar containing ampicillin, and the ampicillin-resistanttransformants were screened by restriction enzyme analysis of theirplasmid DNA to identify the E. coli K12 HB101/pLPCE1A and E. coli K12HB101/pLPCE1A1 transformants. Plasmid DNA was obtained from thetransformants for use in later experiments in substantial accordancewith the procedure of Example 3.

EXAMPLE 14 Construction of Plasmid pBLT

About 1 μg of plasmid pBW32 DNA (FIG. 14, Example 10) in 1 μl of TEbuffer was added to 2 μl of 10× BamHI buffer and 15 μl of H₂O. About 2μl (˜10 units) of restriction enzyme. BamHI were added to the solutionof plasmid pBW32 DNA, and the resulting reaction was incubated at 37° C.for 2 hours. The reaction was stopped by first extracting the reactionmixture with phenol and then extracting the reaction mixture twice withchloroform. About 1 μl of the BamHI-Digested plasmid pBW32 DNA was addedto 1 μl of 10× ligase buffer and 8 μl of H₂O, and after about 1 μl (˜500units) of T4 DNA ligase was added to the solution of DNA, the resultingreaction was incubated at 16° C. overnight.

The ligated DNA constituted the desired plasmid pBW32del, which is about5.6 kb in size and comprises a single HindIII restriction site. Theligated DNA was used to transform E. coli K12 HB101 in substantialaccordance with the procedure of Example 2. The desired E. coli K12HB101/pBW32del transformants were identified by theirampicillin-resistant phenotype and by restriction enzyme analysis oftheir plasmid DNA. Plasmid pBW32del DNA was obtained from thetransformants for use in subsequent constructions in substantialaccordance with the procedure of Example 3.

About 1 μg of plasmid pBW32del in 1 μl of TE buffer was added to 2 μl of10× HindIII buffer and 15 μl of H₂O. About 2 μl (˜10 units) ofrestriction enzyme HindIII were added to the solution of plasmidpBW32del DNA, and the resulting reaction was incubated at 37° C. for 2hours. The sample was diluted to 100 μl with TE buffer and treated withcalf-intestinal alkaline phosphatase in substantial accordance with theprocedure described in Example 2. The reaction was extracted twice withphenol then once with chloroform. The HindIII-digested plasmid pBW32delDNA was then precipitated with ethanol and resuspended in 10 μl of H₂O.

Plasmid pBa18cat (Example 17) was digested with restriction enzymeHindIII, and the ˜0.65 kb HindIII restriction fragment that comprisesthe modified BK enhancer-adenovirus 2 late promoter cassette wasisolated and prepared for ligation in substantial accordance with theprocedure of Example 5. About 0.1 μg of the ˜0.65 kb HindIII restrictionfragment of plasmid pBa18cat in 5 μl of TE buffer was added to 3 μl ofthe solution of HindIII-digested plasmid pBW32del. About 1 μl (˜500units) of T4 DNA ligase and 1 μl of 10× ligase buffer were added to themixture of DNA, and the resulting reaction was incubated at 16° C.overnight.

The ligated DNA constituted the desired plasmid pBLT. A restriction siteand function map of plasmid pBLT is presented in FIG. 18 of theaccompanying drawings. The ligated DNA was used to transform E. coli K12HB101 in substantial accordance with the procedure of Example 2. Thetransformed cells were plated on L agar containing ampicillin, and theampicillin-resistant E. coli K12 HB101/pBLT transformants wereidentified by restriction enzyme analysis of their plasmid DNA. Becausethe ˜0.65 kb HindIII restriction fragment could insert intoHindIII-digested plasmid pBW32del in either one of two orientations,only one of which yields plasmid pBLT, the orientation of the ˜0.65 kbHindIII restriction fragment had to be determined to identify the E.coli K12 HB101/pBLT transformants. Plasmid pBLT DNA was prepared fromthe transformants for use in subsequent constructions in substantialaccordance with the procedure of Example 3.

EXAMPLE 15 Construction of Plasmids pBLThyg1, pBLThyg2, pBLTdhfr1, andpBLTdhfr2

A. Construction of Plasmids pBLThyg1 and pBLThyg2

About 4 μg of plasmid pBLT DNA in 4 μl of TE buffer were added to 2 μlof 10× BamHI buffer and 12 μl of H₂O. About 2 μl (˜10 units) ofrestriction enzyme BamHI were added to the solution of plasmid pBLT DNA,and the resulting reaction was incubated at 37° C. for 2 hours. Thereaction was stopped by extracting the reaction mixture first withphenol and then with chloroform. The BamHI-digested plasmid pBLT DNA wasthen precipitated with ethanol and resuspended in 2 μl of TE buffer.

About 10 μg of plasmid pSV2hyg in 10 μl of TE buffer were added to 10 μlof 10× BamHI buffer and 75 μl of H₂O. About 5 μl (˜25 units) ofrestriction enzyme BamHI were added to the solution of plasmid pSV2hygDNA, and the resulting reaction was incubated at 37° C. for 2 hours. TheBamHI-digested plasmid pSV2hyg DNA was precipitated with ethanol,resuspended in 10 μl of TE buffer, loaded onto an agarose gel, andelectrophoresed until the ˜2.5 kb BamHI restriction fragment thatcomprises the hygromycin resistance-conferring gene was separated fromthe other digestion products. The ˜2.5 kb restriction fragment was thenisolated from the gel and prepared for ligation in substantialaccordance with the procedure of Example 4A; about 2 μg of the desiredfragment were obtained and suspended in 10 μl of TE buffer.

About 2 μl of the BamHI-digested plasmid pBLT DNA and 1 μl of the ˜2.5kb BamHI restriction fragment of plasmid pSV2hyg were added to 1 μl of1033 ligase buffer, 5 μl of H₂O, and 1 μl (˜500 units) of T4 DNA ligase,and the resulting reaction was incubated at 16° C. overnight. Theligated DNA constituted the desired plasmids pBLThyg1 and pBLThyg2. Arestriction site and function map of plasmid pBLThyg1 is presented inFIG. 19 of the accompanying drawings. Plasmids pBLThyg1 and pBLThyg2differ only with respect to the orientation of the ˜2.5 kb BamHIrestriction fragment that encodes the hygromycin resistance-conferringgene.

The ligated DNA was used to transform E. coli K12 HB101 in substantialaccordance with the procedure of Example 2. The transformed cells wereplated onto L agar containing ampicillin, and the ampicillin-resistantE. coli K12 HB101/pBLThyg1 and E. coli K12 HB101/pBLThyg2 transformantswere identified by restriction enzyme analysis of their plasmid DNA.

B. Construction of Plasmids pBLTdhfr1 and pBLTdhfr2

About 100 μg of plasmid pBW32 in 100 μl of TE buffer were added to 15 μlof 10× BamHI buffer and 25 μl of H₂O. About 10 μl (˜50 units) ofrestriction enzyme BamHI were added to the solution of plasmid pBW32DNA, and the resulting reaction was incubated at 37° C. for 2 hours. TheBamHI-digested plasmid pBW32 DNA was precipitated with ethanol,resuspended in 10 μl of TE buffer, loaded onto an agarose gel, andelectrophoresed until the ˜1.9 kb BamHI restriction fragment thatcomprises the dihydrofolate reductase gene was separated from the otherdigestion products. The ˜1.9 kb restriction fragment was then isolatedfrom the gel and prepared for ligation in substantial accordance withthe procedure of Example 4A; about 10 μg of the desired fragment wereobtained and suspended in 50 μl of TE buffer.

About 2 μl of the BamHI-digested plasmid pBLT DNA prepared in Example15A and 1 μl of the ˜1.9 kb BamHI restriction fragment of plasmid pBW32were added to 1 μl of 10× ligase buffer, 5 μl of H₂O, and 1 μl (˜500units) of T4 DNA ligase, and the resulting reaction was incubated at 16°C. overnight. The ligated DNA constituted the desired plasmids pBLTdhfr1and pBLTdhfr2. A restriction site and function map of plasmid pBLTdhfr1presented in FIG. 20 of the accompanying drawings. Plasmids pBLTdhfr1and pBLTdhfr2 differ only with respect to the orientation of the ˜1.9 kbBamHI restriction fragment that encodes the dhfr gene.

The ligated DNA was used to transform E. coli K12 HB101 in substantialaccordance with the procedure of Example 2. The transformed cells wereplated onto L agar containing ampicillin, and the ampicillin-resistantE. coli K12 HB101/pBLTdhfr1 and E. coli K12 HB101/pBLTdhfr2transformants were identified by restriction enzyme analysis of theirplasmid DNA.

EXAMPLE 16 Construction of Plasmids phdTPA and phdMTPA

A. Construction of Intermediate Plasmid pTPA602

About 50 μg of plasmid pTPA103 (Example 10, FIG. 14) in 45 μl ofglass-distilled H₂O were added to 30 μl of 10× EcoRI buffer and 225 μlof H₂O. About 10 μl (˜80 units) of restriction enzyme EcoRI were addedto the solution of plasmid pTPA103 DNA, and the resulting reaction wasincubated at 37° C. for 90 minutes. The EcoRI-digested plasmid pTPA103DNA was precipitated with ethanol, resuspended in 50 μl of 1× loadingbuffer (10% glycerol and 0.02% bromophenol blue), loaded onto an agarosegel, and electrophoresed until the ˜1.1 kb EcoRI restriction fragmentwas separated from the other reaction products. The ˜1.1 kb EcoRIrestriction fragment that comprises the TPA amino-terminal-encoding DNAand was isolated from the gel by electrophoresing the fragment into adialysis bag. The fragment was then precipitated with ethanol andresuspended in 160 μl of H₂O.

About 40 μl of 10× HgsI buffer (0.5M NaCl; 60 mM Tris-HCl, pH=7.4; and0.1M MgCl₂), 200 μl of glass-distilled H₂O, and 20 μl (about 10 units)of restriction enzyme HgaI were added to the solution of ˜1.1 kb EcoRIrestriction fragment, and the resulting reaction was incubated at 37° C.for 4 hours. The HgaI-digested DNA was precipitated with ethanol andthen electrophoresed on a 5% acrylamide gel, and the ˜520 bp restrictionfragment that encodes the amino terminus of TPA was isolated onto DE81paper and recovered. About 5 μg of the ˜520 bp HgaI fragment wereobtained and suspended in 50 μl of H₂O.

About 12.5 μl of 10× Klenow buffer (0.5M Tris-HCl, pH=7.4, and 0.1MMgCl₂), 2 μl of a solution that was 6.25 mM in each of the fourdeoxynucleotide triphosphates, 2 μl of 0.2M DTT, 1 μl of 7 μg/ml BSA,57.5 μl of glass-distilled H₂O, and 2 μl (˜10 units) of Klenow enzyme(Boehringer-Mannheim Biochemicals, 7941 Castleway Dr., P.O. Box 50816,Indianapolis, Ind. 46250) were added to the solution of the ˜520 bp HgaIrestriction fragment, and the resulting reaction was incubated at 20° C.for 30 minutes. The Klenow-treated DNA was incubated at 70° C. for 15minutes and precipitated with ethanol.

About 500 picomoles of BamHI linker (5′-CGGGATCCCG-3′, double-strandedand obtained from New England Biolabs) were phosphorylated usingpolynucleotide kinase in a total reaction volume of 25 μl. The reactionwas carried out in substantial accordance with the procedure describedin Example 6A. The kinased BamHI linkers were added to the solution ofKlenow-treated, ˜520 bp HgaI restriction fragment together with 15 μl of10× ligase buffer, 7 μl (˜7 Weiss units) of T4 DNA ligase, and enoughglass-distilled H₂O to bring the reaction volume to 150 μl. Theresulting reaction was incubated at 16° C. overnight.

The ligation reaction was heat-inactivated, and the DNA was precipitatedwith ethanol and resuspended in 5 μl of 10× BamHI buffer and 45 μl ofH₂O. About 1 μl (˜16 units) of restriction enzyme BamHI was added to thesolution of DNA, and the resulting reaction was incubated at 37° C. for90 minutes. Then, another 16 units of BamHI enzyme were added to thereaction mixture, and the reaction was incubated at 37° C. for another90 minutes. The reaction mixture was then electrophoresed on a 5%polyacrylamide gel, and the ˜530 bp HgaI restriction fragment, now withBamHI ends, was purified from the gel in substantial accordance with theprocedure of Example 6A. About 2 μg of the desired fragment wereobtained and suspended in 20 μl of H₂O.

BamHlI-digested, dephosphorylated plasmid pBR322 DNA can be obtainedfrom New England Biolabs. About 0.1 μg of BamHI-digested,dephosphorylated plasmid pBR322 in 2 μl of H₂O was added to 1 μl of the˜530 bp HgaI restriction fragment, with BamHI ends, of plasmid pTPA103,14 μl of H₂O, and 1 μl (˜1 Weiss unit) of T4 DNA ligase, and theresulting reaction was incubated at 16° C. overnight. The ligated DNAconstituted the desired plasmid pTPA602 and an equivalent plasmiddesignated pTPA601, which differs from plasmid pTPA602 only with respectto the orientation of the inserted, ˜530 bp restriction fragment. Arestriction site and function map of plasmid pTPA602 is presented inFIG. 21 of the accompanying drawings.

The ligated DNA was used to transform E. coli K12MM294 in substantialaccordance with the procedure of Example 2, except that 50 mM CaCl₂ wasused in the procedure. The transformed cells were plated on L agarcontaining ampicillin, and the ampicillin-resistant E. coliK12MM294/pTPA602 and E. coli K12MM294/pTPA601 cells were identified byrestriction enzyme analysis of their plasmid DNA. Presence of an ˜530 bpBamHI restriction fragment indicated that the plasmid was either pTPA602or plasmid pTPA601.

B. Construction of Intermediate Plasmid pTPA603

About 5 μg of plasmid pTPA602 were dissolved in 20 μl of 10× BglII and180 μl of H₂O. About 3 μl (˜24 units) of restriction enzyme BglII wereadded to the solution of plasmid pTPA602 DNA, and the resulting reactionwas incubated at 37° C. for 90 minutes. Then, ˜13 μl of 10× BamHI bufferwere added to the reaction mixture to bring the salt concentration ofthe reaction mixture up to that recommended for SalI digestion, and 2 μl(˜20 units) of restriction enzyme SalI were added to the reaction. Thereaction was incubated at 37° C. for another 2 hours; then, the DNA wasprecipitated with ethanol, resuspended in 75 μl of loading buffer,loaded onto an agarose gel, and electrophoresed until the ˜4.2 kbBglII-SalI restriction fragment was separated from the other digestionproducts. The region of the gel containing the ˜4.2 kb BglII-SalIrestriction fragment was excised from the gel, frozen, and the frozensegment was wrapped in plastic and squeezed to remove the ˜4.2 kbfragment. The DNA was precipitated and resuspended in 20 μl of H₂O;about 200 nanograms of the desired fragment were obtained.

About 12 μg of plasmid pTPA103 were dissolved in 15 μl of 10× BglIIbuffer and 135 μl of H₂O. About 2 μl (˜16 units) of restriction enzymeBglII were added to the solution of plasmid pTPA103 DNA, and theresulting reaction was incubated at 37° C. for 90 minutes. About 10 μlof 10× BamHI buffer were added to the solution of BglII-digested plasmidpTPA103 DNA to bring the salt concentration of the reaction mixture upto that required for SalI digestion. Then, about 2 μl (˜20 units) ofrestriction enzyme SalI were added to the solution of BglII-digestedplasmid pTPA103 DNA, and the reaction was incubated at 37° C. foranother 90 minutes. The BglII-SalI digested plasmid pTPA103 DNA wasconcentrated by ethanol precipitation and then loaded onto an agarosegel, and the ˜2.05 kb BglII-SalI restriction fragment that encodes allbut the amino-terminus of TPA was isolated from the gel, precipitatedwith ethanol and resuspended in 20 μl of H₂O. About 2 μg of the desiredfragment were obtained.

About 5 μl of the ˜4.2 kb BglII-SalI restriction fragment of plasmidpTPA602 and 2 μl of the ˜2.05 kb BglII-SalI restriction fragment ofplasmid pTPA103 were added to 2 μl of 10× ligase buffer, 10 μl of H₂O,and 1 μl (˜1 Weiss unit) of T4 DNA ligase, and the resulting ligationreaction was incubated at 16° C. overnight. The ligated DNA constitutedthe desired plasmid pTPA603. A restriction site and function map ofplasmid pTPA603 is presented in FIG. 22 of the accompanying drawings.

The ligated DNA was used to transform E. coli K12MM294 in substantialaccordance with the procedure of Example 2, except that 50 mM CaCl₂ wasused in the procedure. The transformed cells were plated on L agarcontaining ampicillin, and the ampicillin-resistant E. coliK12MM294/pTPA603 transformants were identified by restriction enzymeanalysis of their plasmid DNA.

C. Construction of Plasmid pMTPA603

About 100 μg of plasmid pBLT (Example 14, FIG. 18) in 100 μl of TEbuffer were added to 10 μl of 10× SstI (SstI is equivalent torestriction enzyme SacI) buffer (60 mM Tris-HCl, pH=7.4; 60 mM MgCl₂; 60mM 2-mercaptoethanol; and 1 mg/ml BSA) and 25 μl of H₂O. About 10 μl(˜50 units) of restriction enzyme SstI were added to the solution ofplasmid pBLT DNA, and the resulting reaction was incubated at 37° C. for2 hours. The SstI-digested plasmid pBLT DNA was precipitated withethanol and resuspended in 10 μl of 10× BglII buffer and 85 μl of H₂O.About 5 μl (˜50 units) of restriction enzyme BglII were added to thesolution of SstI-digested plasmid pBLT DNA, and the resulting reactionwas incubated at 37° C. for 2 hours.

The BglII-SstI-digested plasmid pBLT DNA was precipitated with ethanol,resuspended in 10 μl of H₂O, loaded onto an agarose gel,electrophoresed, and the ˜690 bp BglII-SstI restriction fragment, whichcontains that portion of the modified TPA coding sequence wherein thedeletion to get the modified TPA coding squence has occurred, of plasmidpBLT was isolated from the gel in substantial accordance with theprocedure of Example 4A. About 5 μg of the desired ˜690 bp BglII-SstIrestriction fragment of plasmid pBLT was obtained and suspended in 100μl of H₂O.

About 5 μg of plasmid pTPA603 (Example 16B, FIG. 22) in 5 μl of TEbuffer were added to 10 μl of 10× SstI buffer and 95 μl of H₂O. About 5μl (˜50 units) of restriction enzyme SstI were added to the solution ofplasmid pTPA603 DNA, and the resulting reaction was incubated at 37° C.for 2 hours. The SstI-digested plasmid pTPA603 DNA was precipitated withethanol and resuspended in 10 μl of 10× BglII buffer and 85 μl of H₂O.About 5 μl (˜50 units) of restriction enzyme BglII were added to thesolution of SstI-digested plasmid pTPA603 DNA, and the resultingreaction was incubated at 37° C. for 2 hours. The BglII-SstI-digestedplasmid pTPA603 DNA was diluted to 100 μl in TE buffer and treated withcalf-intestinal alkaline phosphatase in substantial accordance with theprocedure of Example 2. The DNA was then precipitated with ethanol andresuspended in 10 μl of H₂O.

About 5 μl of the BglII-SstI-digested plasmid pTPA603 and 2 μl of the˜690 bp BglII-SstI restriction fragment of plasmid pBLT were added to 2μl of 10× ligase buffer, 10 μl of H₂O, and 1 μl (˜1000 units) of T4 DNAligase, and the resulting ligation reaction was incubated at 16° C.overnight. The ligated DNA constituted the desired plasmid pMTPA603.Plasmid pMTPA603 is thus analogous in structure to plasmid pTPA603 (FIG.22), except that plasmid pMTPA603 encodes modified TPA, and plasmidpTPA603 encodes TPA.

The ligated DNA was used to transform E. coli K12 HB101 in substantialaccordance with the procedure of Example 2. The transformed cells wereplated on L agar containing ampicillin, and the ampicillin-resistant E.coli K12 HB101/pMTPA603 transformants were identified by restrictionenzyme analysis of their plasmid DNA.

D. Construction of Plasmid phdTPA

About 10 μg of plasmid pTPA603 (Example 16B, FIG. 22) in 10 μl of TEbuffer were added to 10 μl of 10× BamHI buffer and 85 μl of H₂O. About 5μl (˜50 units) of restriction enzyme BamHI were added to the solution ofplasmid pTPA603 DNA, and the resulting reaction was incubated at 37° C.for 2 hours. The BamHI-digested plasmid pTPA603 DNA was precipitatedwith ethanol, resuspended in 10 μl of H₂O, loaded onto an agarose gel,and electrophoresed until the ˜1.90 kb BamHI restriction fragment thatencodes TPA was separated from the other digestion products. The ˜1.90kb BamHI restriction fragment was isolated from the gel and resuspendedin 50 μl of TE buffer; about 4 μg of the desired fragment were obtained.

About 2 μg of plasmid phd (Example 12, FIG. 16) in 2 μl of TE bufferwere added to 2 μl of 10× BclI buffer and 14 μl of H₂O. About 2 μl (˜10units) of restriction enzyme BclI were added to the solution of plasmidphd DNA, and the resulting reaction was incubated at 50° C. for 2 hours.The reaction was stopped by extracting the reaction mixture first withphenol and then twice with chloroform. The BclI-digested plasmid phd DNAwas then precipitated with ethanol and resuspended in 20 μl of TEbuffer.

About 1 μl of the BclI-digested plasmid phd and 2 μl of the ˜1.90 kbBamHI restriction fragment of plasmid pTPA603 were added to 1 μl of 10×ligase buffer, 5 μl of H₂O, and 1 μl (˜500 units) of T4 DNA ligase. Theresulting ligation reaction was incubated at 16° C. overnight. Theligated DNA constituted the desired plasmid phdTPA. A restriction siteand function map of plasmid phdTPA is presented in FIG. 23 of theaccompanying drawings.

The ligated DNA was used to transform E. coli K12 HB101 (NRRL B-15626)in substantial accordance with the procedure of Example 2. Thetransformation mixture was plated on L agar containing ampicillin, andthe ampicillin-resistant E. coli K12 HB101/phdTPA cells were identifiedby restriction enzyme analysis. The ˜1.90 kb BamHI restriction fragmentcould insert into BclI-digested plasmid phd in either one of twoorientations, only one of which places the TPA coding sequence in theproper position to be expressed under the control of the BKenhancer-adenovirus late promoter cassette and thus results in thedesired plasmid phdTPA.

E. Construction of Plasmid phdMTPA

About 10 μg of plasmid pMTPA603 (Example 16C) in 10 μl of TE buffer wereadded to 10 μl of 10× BamHI buffer and 85 μl of H₂O. About 5 μl (˜50units) of restriction enzyme BamHI were added to the solution of plasmidpMTPA603 DNA, and the resulting reaction was incubated at 37° C. for 2hours. The BamHI-digested plasmid pMTPA603 DNA was precipitated withethanol, resuspended in 10 μl of H₂O, loaded onto an agarose gel, andelectrophoresed until the ˜1.35 kb BamHI restriction fragment thatencodes modified TPA was separated from the other digestion products.The ˜1.35 kb BamHI restriction fragment was isolated from the gel andresuspended in 20 μl of TE buffer; about 4 μg of the desired fragmentwere obtained.

About 1 μl of the BclI-digested plasmid phd prepared in Example 16D and2 μl of the ˜1.35 kb BamHI restriction fragment of plasmid pMTPA603 wereadded to 1 μl of 10× ligase buffer, 5 μl of H₂O, and 1 μl (˜500 units)of T4 DNA ligase. The resulting ligation reaction was incubated at 16°C. overnight. The ligated DNA constituted the desired plasmid phdMTPA. Arestriction site and function map of plasmid phdMTPA is presented inFIG. 24 of the accompanying drawings.

The ligated DNA was used to transform E. coli K12 HB101 in substantialaccordance with the procedure of Example 2. The transformation mixturewas plated on L agar containing ampicillin, and the ampicillin-resistantE. coli K12 HB101/phdMTPA cells were identified by restriction enzymeanalysis of their plasmid DNA. The ˜1.35 kb BamHI restriction fragmentcould insert into BclI-digested plasmid phd in either one of twoorientations, only one of which places the TPA coding sequence in theproper position to be expressed under the control of the BKenhancer-adenovirus late promoter and thus results in the desiredplasmid phdMTPA.

EXAMPLE 17 Construction of an Improved BK Enhancer-Adenovirus LatePromoter Cassette

The transcription-enhancing effect of the BK enhancer can besignificantly increased by placing the enhancer from 0 to 300nucleotides upstream of the 5′ end of the CAAT region or CAAT regionequivalent of an adjacent eukaryotic promoter. The sequence andfunctional elements of the present BK enhancer-adenovirus 2 latepromoter cassette, before modification to achieve greater enhancingactivity, is depicted below. This depiction assumes that the BK enhanceris from the prototype strain of BK virus, available from the ATCC underthe VR-837. However, ATCC VR-837 consists of a mixture of BK variants.Plasmid pBa18cat and the other BK enhancer-containing plasmids of theinvention comprise this BK enhancer variant and not the BK prototypeenhancer depicted below. As stated above, however, any BK enhancervariant can be used in the methods and compounds of the presentinvention. Plasmid pBa18cat can be obtained in E. coli K12 HB101 cellsfrom the Northern Regional Research Center, Peoria, Ill. 61604 under theaccession number NRRL B-18267.

   HinIII                                                          605′-AAGCTTTTCT CATTAAGGGA AGATTTCCCC AGGCAGCTCT TTCAAGGCCT AAAAGGTCCA   ------                                                                   120   TGAGCTCCAT GGATTCTTCC CTGTTAAGAA CTTTATCCAT TTTTGCAAAA ATTGCAAAAG                                  StuI                             180   AATAGGGATT TCCCCAAATA GTTTTGCTAG GCCTCAGAAA AAGCCTCCAC ACCCTTACTA                                 -------                                                                   240   CTTGAGAGAA AGGGTGGAGG CAGAGGCGGC CTCGGCCTCT TATATATTAT AAAAAAAAAG     *--------------------- first repeat of the BK enhancer-------- 300   GCCACAGGGA GGAGCTGCTT ACCCATGGAA TGCAGCCAAA CCATGACCTC AGGAAGGAAA                                                                   360   ---------* *----------second repeat of the BK enhancer----------*   GTGCATGACT CACAGGGGAA TGCAGCCAAA CCATGACCTC AGGAAGGAAA GTGCATGACT                                                                   420   *------------------------------ third repeat of the BK enhancer--   CACAGGGAGG AGCTGCTTAC CCATGGAATG CAGCCAAACC ATGACCTCAG GAAGGAAAGT   ------*|------43 bp insert, not found in BK(DUN)-------|        480   GCATGACTGG GCAGCCAGCC AGTGGCAGTT AATAGTGAAA CCCCGCCGAC AGACATGTTT                                                                   540   TGCGAGCCTA GGAATCTTGG CCTTGTCCCC AGTTAAACTG GACAAAGGCC ATGGTTCTGC   StuI PvuII                 SstI                                 600   GCCAGGCTGT CCTCGAGCGG TGTTCCGCGG TCCTCCTCGT ATAGAAACTC GGACCACTCT      ------                 ------                                                                   660   GAGACGAAGG CTCGCGTCCA GGCCAGCACG AAGGAGGCTA AGTGGGAGGG GTAGCGGTCG                                                                   720   TTGTCCACTA GGGGGTCCAC TCGCTCCAGG GTGTGAAGAC ACATGTCGCC CTCTTCGGCA                                    CAAT Region                    780   TCAAGGAAGG TGATTGGTTT ATAGGTGTAG GCCACGTGAC CGGGTGTTCC TGAAGGGGGG                                      start site of transcription    TATA Box                          *--->                        840   CTATAAAAGG GGGTGGGGGC GCGTTCGTCC TCACTCTCTT CCGCATCGCT GTCTGCGAGG    -------                                     874        BclI linker             HindIII    GCCAGCTGAT CAGCCTAGGC TTTGCAAAAA GCTT-3′

wherein A is deoxyadenyl; G is deoxyguanyl; C is deoxycytidyl; and T isthymidyl.

The prototype BK enhancer is defined by the three repeated sequencesindicated in the sequence above and functions similarly, with respect toan adjacent sequence, in either orientation. To bring the enhancer, morespecifically, the 3′ end of the third repeat (which depends on theorientation) of the BK enhancer, closer to the 5′ end of the CAAT regionof the adenovirus-2 late promoter, about 82 μg of SstI-digested plasmidpBLcat DNA in 170 μl of TE buffer were added to 20 μl of 5× Ba131nuclease buffer (0.1M Tris-HCl, pH=8.1; 0.5M NaCl; 0.06M CaCl₂; and 5 mMNa₂EDTA) and 9 μl of Ba131 nuclease, which was composed of 6 μl (˜6units) of “fast” and 3 μl (˜3 units) of “slow” Ba131 enzyme (marketed byInternational Biotechnologies, Inc., P.O. Box 1565, New Haven, Conn.06506). The reaction was incubated at 30° C. for about 3 minutes; then,after about 10 μl of 0.1M EGTA were added to stop the reaction, theBa131-digested DNA was collected by ethanol precipitation andcentrifugation. The DNA pellet was resuspended in 1× Klenow buffer andtreated with Klenow enzyme in substantial accordance with procedurespreviously described herein.

The Klenow-treated DNA was resuspended in 10 μl of TE buffer; about 1 μlof the DNA was then self-ligated in 10 μl of 1× ligase buffer using T4DNA and RNA ligase as previously described. The ligated DNA was used totransform E. coli K12 HB101, and then the transformants were plated ontoL agar containing ampicillin. Restriction enzyme analysis was used todetermine which transformants contained plasmids with anappropriately-sized BK enhancer-adenovirus 2 late promoter cassette. Theforegoing procedure generates a number of plasmids in which the BKenhancer is placed within 0 to 300 nucleotides upstream of the CAATregion of the adenovirus late promoter. One plasmid resulting from theabove procedure was designated plasmid pBa18cat. Plasmid pBa18cat isavailable from the NRRL under the accession number NRRL B-18267. PlasmidpBa18cat contains a variant of the BK enhancer that is believed tocontain two repeat sequences of about 90 bp each. This variant enhancercan be used in the method of the present invention by placing the 3′ endof the second repeat within 0 to 300 nucleotides of the CAAT region ofthe adenovirus late promoter.

Those skilled in the art will recognize that the foregoing procedureproduced a number of distinct plasmids, of which plasmid pBa18cat isillustrative. These plasmids, as a group, represent placing the BKenhancer at a variety of distances less than 300 nucleotides from theCAAT region of the Ad2 late promoter and thus comprise an importantaspect of the present invention. This method for improving the activityof a BK enhancer, which can be achieved using the foregoing procedure orothers known to those skilled in the art, can be used with any BKenhancer and any eukaryotic promoter.

EXAMPLE 18 Construction of Eukaryotic Host Cell Transformants of theExpression Vectors of the Present Invention and Determination ofRecombinant Gene Expression Levels in Those Transformants

An important aspect of the present invention concerns the use of the BKenhancer to stimulate gene expression in the presence of the E1A geneproduct. Because 293 cells constitutively express the E1A gene product,293 cells are the preferred host for the eukaryotic expression vectorsof the present invention. 293 cells are human embryonic kidney cellstransformed with adenovirus type 5 (note that any particular type ofadenovirus can be used to supply the E1A gene product in the method ofthe present invention) and are available from the ATCC under theaccession number CRL 1573. However, the expression vectors of thepresent invention function in a wide variety of host cells, even if theE1A gene product is not present. Furthermore, the E1A gene product canbe introduced into a non-E1A-producing cell line either bytransformation with a vector of the present invention that comprises theE1A gene, such as plasmids pLPCE1A and pLPCE1A1, or with sheeredadenovirus DNA, or by infection with adenovirus.

The transformation procedure described below refers to 293 cells as thehost cell line; however, the procedure is generally applicable to mosteukaryotic cell lines. A variety of cell lines have been transformedwith the vectors of the present invention; some of the actualtransformants constructed and related information are presented in theTables accompanying this Example. Because of the great number ofexpression vectors of the present invention, the transformationprocedure is described generically, and the actual transformantsconstructed are presented in the Tables.

293 cells are obtained from the ATCC under the accession number CRL 1573in a 25 mm² flask containing a confluent monolayer of about 5.5×10⁶cells in Eagle's Minimum Essential Medium with 10% heat-inactivatedhorse serum. The flask is incubated at 37° C.; medium is changed twiceweekly. The cells are subcultured by removing the medium, rinsing withHank's Balanced Salts solution (Gibco), adding 0.25% trypsin for 1-2minutes, rinsing with fresh medium, aspirating, and dispensing into newflasks at a subcultivation ratio of 1:5 or 1:10.

One day prior to transformation, cells are seeded at 0.7×10⁶ cells perdish. The medium is changed 4 hours prior to transformation. Sterile,ethanol-precipitated plasmid DNA dissolved in TE buffer is used toprepare a 2× DNA-CaCl₂ solution containing 40 μg/ml DNA and 250 mMCaCl₂, 2× HBS is prepared containing 280 mM NaCl, 50 mM Hepes, and 1.5mM sodium phosphate, with the pH adjusted to 7.05-7.15. The 2× DNA-CaCl₂solution is added dropwise to an equal volume of sterile 2× HBS. A oneml sterile plastic pipette with a cotton plug is inserted into themixing tube that contains the 2× HBS, and bubbles are introduced byblowing while the DNA is being added. The calcium-phosphate-DNAprecipitate is allowed to form without agitation for 30-45 minutes atroom temperature.

The precipitate is then mixed by gentle pipetting with a plasticpipette, and one ml (per plate) of precipitate is added directly to the10 ml of growth medium that covers the recipient cells. After 4 hours ofincubation at 37° C., the medium is replaced with DMEM with 10% fetalbovine serum and the cells allowed to incubate for an additional 72hours before providing selective pressure. For transformants expressingrecombinant human protein C, the growth medium contained 1 to 10 μg/mlvitamin K, a cofactor required for γ-carboxylation of the protein. Forplasmids that do not comprise a selectable marker that functions ineukaryotic cells, the transformation procedure utilizes a mixture ofplasmids: the expression vector of the present invention that lacks aselectable marker; and an expression vector that comprises a selectablemarker that functions in eukaryotic cells. This co-transformationtechnique allows for the identification of cells that comprise both ofthe transforming plasmids.

For cells transfected with plasmids containing the hygromycinresistance-conferring gene, hygromycin is added to the growth medium toa final concentration of about 200 to 400 μg/ml. The cells are thenincubated at 37° C. for 2-4 weeks with medium changes at 3 to 4 dayintervals. The resulting hygromycin-resistant colonies are transferredto individual culture flasks for characterization. The selection ofneomycin (G418 is also used in place of neomycin)-resistant colonies isperformed in substantial accordance with the selection procedure forhygromycin-resistant cells, except that G418 is added to a finalconcentration of 400 μg/ml rather than hygromycin. 293 cells are dhfrpositive, so 293 transformants that contain plasmids comprising the dhfrgene are not selected solely on the basis of the dhfr-positivephenotype, which is the ability to grow in media that lacks hypoxanthineand thymine. Cell lines that do lack a functional dhfr gene and aretransformed with dhfr-containing plasmids can be selected for on thebasis of the dhfr+ phenotype.

The use of the dihydrofolate reductase (dhfr) gene as a selectablemarker for introducing a gene or plasmid into a dhfr-deficient cell lineand the subsequent use of methotrexate to amplify the copy number of theplasmid has been well established in the literature. Although the use ofdhfr as a selectable and amplifiable marker in dhfr-producing cells hasnot been well studied, efficient coamplification in primate cellsrequires an initial selection using a directly selectable marker beforethe coamplification using methotrexate. The use of the present inventionis not limited by the selectable marker used. Moreover, amplifiablemarkers such as metallothionein genes, adenosine deaminase genes, ormembers of the multigene resistance family, exemplified byP-glycoprotein, can be utilized. In 293 cells, it is advantageous totransform with a vector that contains a selectable marker such as thehygromycin B resistance-conferring gene and then amplify usingmethotrexate, which cannot be used for direct selection of murinedhfr-containing plasmids in 293 cells. The levels of coamplification canbe measured using Southern hybridization or other methods known in theart. Tables 7 and 8 display the results of coamplification experimentsin 293 cells.

TABLE 3 Expression Levels in 293 Cell Transformants Expressed ExpressionLevel (as measured by amount Plasmid Gene of expressed gene product incell media) pLPChyg1 Protein C 0.1-4.0 μg/10⁶ cells/day. pLPCdhfr1Protein C 0.1-4.0 μg/10⁶ cells/day. pLPC4 Protein C 0.1-2.0 μg/10⁶cells/day, cotransformed with plasmid pSV2hyg pLPC5 Protein C 0.1-2.0μg/10⁶ cells/day, cotransformed with plasmid pSV2hyg pLPChd1 Protein C˜1.2 μg/10⁶ cells/day, phdTPA TPA in a transient assay conducted 24-36hours post-transformation, about 0.5- 1.25 μg/10⁶ cells, if the VA geneproduct is present in the host cell and about 10-fold less if not.Stable transformants produce about 2.5-3.8 μg/ 4 × 10⁶ cells/day.

TABLE 4 Expression Levels in MK2 (ATCC CCL7) Cell Transformants PlasmidExpressed Gene Expression Level pLPChyg1 Protein C 0.005-0.040 μg/10⁶cells/day. pLPChd Protein C 0.025-0.4 μg/10⁶ cells/day. pLPC4 Protein C0.025-0.15 μg/10⁶ cells/day, cotransformed with plasmid pSV2hyg. pLPC5Protein C 0.025-0.18 μg/10⁶ cells/day, cotransformed with plasmidpSV2hyg.

TABLE 5 Relative Levels of Chloramphenicol Acetylantransferase (CAT)Produced by Recombinant Plasmids in Various Human and Monkey Cell LinesRelative Level* of CAT in Cell Line: 293 COS-1 (ATCC (ATCC MK2 PlasmidCRL 1573) k816-4** CRL 1650) (ATCC CCL7) pLPcat 0.17 0.16 0.18 0.06pSV2cat 1 1 1 1 pBLcat 10.4 2.7 1.4 1.3 pSBLcat 3.9 5.4 3.4 2.8 pSLcat0.20 3.6 NT 1.05 pBa18cat 17 1.8 NT 1.2 *The values for the relativelevels of CAT produced in each cell line were based on the level of CATfrom plasmid pSV2cat as unity in that cell line. Results are the averageof from 2 to 6 individual determinations of each data point. ND = notdetected. NT = not tested. Plasmid pSLcat is analogous to plasmid pBLcatbut has the SV40 enhancer rather than the BK enhancer. Only the 293 cellline produces E1A. The COS and k816-4 cell lines produce T antigen.**k816-4 cells were prepared by transformation of primary human kidneycells with a plasmid, designated pMK16,8-16 (obtained from Y. GluzmanCold Spring Harbor), containing an SV40 genome with a defect in theorigin of replication. This cell line constitutively produces the Tantigen of SV40. The k816-4 cell line is essentially the same as cellline SV1, an SV40-transformed human kidney line, described by E. O.Major, Polyomaviruses and Human Neurological Disease (Alan R. Liss,Inc., N.Y. 1983, # cds D. Madden, and J. Sever).

TABLE 6 Relative Levels of Chloramphenicol Acetylatransferase (CAT)Produced by Recombinant Plasmids in Various Human and Monkey Kidney CellLines Corrected for Relative Differences in Plasmid Copy Number RelativeLevel* of CAT in Cell Line: Plasmid 293 k816-4 MK2 pLPcat 0.18 0.250.015 pSV2cat 1 2.1 0.25 pBcat 12.6 5.8 0.32 *The values for therelative levels of CAT produced in each cell line were corrected bydividing the level of CAT in the cell lysate by the amount of plasmidDNA, as determined by hybridization analysis, in the same cell lysate.The corrected value for plasmid pSV2cat in 293 cells was taken as unity.

TABLE 7 Methotrexate sensitivity and level of HPC expression from 293cells transformed by plasmid pLPChd and initially selected forhygromycin resistance. Level methotrexatc Number of Level of HPC (μM)colonies (ng/10⁶ cells) 0 confluent 575 0.05 confluent 1794 0.2 500+3786 0.4 32 235 0.8 53 325 1.6 58 165 3.2 44 310

TABLE 8 Level of HPC in clones selected for growth in increasing levelsof methotrexate following initial selection with hygromycin (A) orG418(B) HPC (ng/10⁶ cells/day) in MTS (μM) level of: 0.05 0.1 0.2 0.40.8 1.6 3.2 5.0 10 Clone A Pool 270 210 160 290 1118 −1 1820 310 370 150350 360 −10 2170 220 370 110 200 −35 1520 210 200 −26 1300 240 460 150160 −37 2400 470 630 530 580 −21 1100 1700 3100 2450 2060 1100 680 21subclones 21-1 4100 21-2 4300 21-3 3010 21-4 2970 21-5 4130 21-6 283021-7 1130 21-10b-1 5790 21-10-3 4700 21-10b-3 12175 21-10b-4 1115521-10b-5 10235 21-10b-6 8490 21-10-7 <20 21-10b-7 4990 21-10b-10 950021-10-2 1705 B* Pool 0925 315 600 2200 Subclones hdA6 37000 hdA4 22250A1 40000 A2 33750 A3 44250 *denotes cotransfections with plsamids pLPChdand pSV2neo.

EXAMPLE 19

Cell line AV12 (ATCC CRL 9595) can be transformed in substantialaccordance with the procedure described for 293 cells in Example 18.However, unlike 293 cells, AV12 cells can be directly selected withmethotrexate (200-500 nM) when transformed with a vector containing themurine dhfr gene. Table 6, below, illustrates the advantages ofproducing a γ-carboxylated protein, in this instance, activated humanprotein C, in an adenovirus-transformed host cell. The transformantswere selected using hygromycin B or methotrexate; transformants produced˜2 to 4 μg/ml of human protein C. Protein C levels can be increased to˜10 μg/ml by amplification with methotrexate. The protein C wasactivated and its activity determined as described in Grinnell et al.,1987, Bio/Technology 5:1189. Activity values are based on an activity of1.0 for human plasma protein. The activities are expressed in ratios ofactivated partial thromboplastin time (APTT) over amidolytic (serineprotease) activity or amount of protein C antigen (ELISA).

TABLE 9 Functional Activity of Protein C Produced inAdenovirus-transformed Cell Lines Cell Line APTT/Amidolytic APTT/ELISA293/pLPChd 1.2-1.7 1.2-1.7 AV12/pLPChd  0.9-1.45  0.9-1.45 SA7/pLPChd nd1.0  SV20/pLPChd nd 0.95 nd = not determined; SA7 and SV20 are Syrianhamster cell lines transformed with simian adenovirus 7 and simian virus20, respectively.

Table 9 shows that the recombinant protein C activity produced in anadenovirus-transformed host cell is at least as active as that found inhuman blood. In non-adenovirus-transformed host cells, the anticoagulantactivity of the recombinant protein C produced never exceeds 60% of theactivity of human blood-derived protein C.

EXAMPLE 20 Construction of Plasmids p4-14 and p2-5, Plasmids that Encodethe Tripartite Leader of Adenovirus

Plasmids p4-14 and p2-5 both utilize the improved BK-enhancer adenoviruslate promoter cassette of plasmid pBa18cat and the tripartite leader ofadenovirus to drive high level expression of human protein C ineukaryotic host cells. The DNA encoding the adenovirus tripartite leader(TPL) was isolated from adenovirus; numbers in parentheses afterrestriction enzyme cut sites refer to map units of adenovirus.

Plasmid pUC13 (commercially available from BRL) was digested withrestriction enzymes SphI and BamHI and then ligated with theTPL-encoding ˜7.2 kb SphI (5135)-BclI (12,301) restriction fragment ofadenovirus type 2 to yield plasmid pTPL4. Part of an intron was deletedfrom the TPL-encoding DNA by digesting plasmid pTPL4 with restrictionenzymes SauI (7616) and BglII (8904), treating with Klenow enzyme, andreligating to yield plasmid pATPL. Plasmid PATPL was then digested withrestriction enzyme XhoI, and the ˜2.62 kb XhoI fragment encoding the TPL(XhoI sites at 5799 and 9689 of adenovirus) was isolated and preparedfor ligation.

Plasmid pBLcat was digested with restriction enzymes XhoI and BclI andthen ligated with the linker:

to yield plasmid pBΔLcat. This construction replaces the adenovirus latepromoter on plasmid pBLcat with the linker sequence. Plasmid pBΔLcat wasdigested with restriction enzyme XhoI and ligated with the ˜2.62 kb XhoIrestriction fragment of plasmid pATPL to yield plasmid PBΔL-TPL, inwhich the TPL-encoding fragment is correctly positioned to place the BKenhancer, adenovirus major late promoter, and TPL in alignment forexpression of the CAT gene.

Plasmid p2-5 was then constructed by ligating these fragments: (1) theAatII-BclI restriction fragment of plasmid pLPChd1, which encodes thedhfr gene; (2) the protein C-encoding, BclI restriction fragment ofplasmid pLPChd1; (3) the TPL-encoding PvuII-BclI restriction fragment ofplasmid pBΔL-TPL; and (4) the BK-enhancer-Ad2MLP-encoding PvuII-AatIIrestriction fragment of plasmid pBa18cat. Plasmid p2-5 thus contains thedhfr gene as a selectable, amplifiable marker and the BK enhancer,Ad2MLP, and Ad2TPL correctly positioned to drive expression of humanprotein C.

Plasmid p4-14 is analogous to plasmid p2-5 but was constructed via anintermedite plasmid designated pBa18TPL. Plasmid pBa18TPL wasconstructed by ligating fragments 1, 3, and 4, used in the constructionof plasmid p2-5, as described in the preceding paragraph. PlasmidpBa18TPL was then digested with restriction enzyme XhoI treated withKlenow enzyme to make the XhoI ends blunt-ended and then ligated withthe human protein C-encoding, Klenow-treated BclI restriction fragmentof plasmid pLPChd1 to yield plasmid p4-14. Thus, plasmid p4-14 onlydiffers from plasmid p2-5 in that the protein C-encoding DNA wasinserted at the XhoI site in the fragment derived from plasmid pBΔL-TPL,whereas in plasmid p2-5, this DNA was inserted at the BclI site in theDNA derived from plasmid pBΔL-TPL.

Plasmid p4-14 and p2-5 drive high-level expression of human protein C.In AV12 cells, plasmids p4-14 and p2-5 can be directly selected using200-500 nM methotrexate. AV12/p4-14 transformants, before amplification,express 5-6 times more human protein C than AV12/pLPCdhfr transformants.Amplification with methotrexate further increases the amount of humanprotein C produced by the cells. Plasmids p4-14 and p2-5 are thusillustrative of the higher expression levels achieved using the TPL ofadenovirus.

I claim:
 1. In a method for producing a protein that is naturallyγ-carboxylated, properly folded and processed and wherein said proteinis encoded in a recombinant DNA vector such that said protein isexpressed when a eukaryotic host cell containing said vector is culturedunder suitable expression conditions, the improvement comprising: (a)inserting said vector into an adenovirus-transformed host cell; and (b)culturing said host cell of step a) under growth conditions and in mediacontaining sufficient vitamin K for carboxylation.
 2. The method ofclaim 1 wherein the adenovirus-transformed host cell is selected fromthe group consisting of the human embryonic kidney 293 and the AV12 celllines.
 3. The method of claim 2, wherein said γ-carboxylated protein ishuman protein C.
 4. The method of claim 3, wherein said host cellcultured in step (b) is selected from the group consisting of 293/pLPC,293/pLPC4, 293/pLPChyg1, 293/pLPCdhfr1, 293/pLPChd1, 293/pLPChT1,293/p4-14, AV12/pLPC, AV12/pLPC4, AV12/pLPChyg1, AV12/pLPCdhfr1,AV12/pLPChd1, AV12/pLPChT1, and AV12/p4-14 host cells.
 5. The method ofclaim 1, wherein said vector comprises a BK virus enhancer.
 6. Themethod of claim 5, wherein said host cell expresses an immediate-earlygene product of a DNA virus.
 7. The method of claim 6, wherein saidimmediate-early gene product of a DNA virus is an EIA gene product ofadenovirus.
 8. The method of claim 5, wherein said BK virus enhancer ispresent in tandem with a eukaryotic promoter.
 9. The method of claim 8,wherein said eukaryotic promoter is an adenovirus late promoter (MLP).10. The method of claim 1, wherein said vector comprises a BKenhancer-adenovirus late promoter cassette.
 11. The method of claim 2,wherein the adenovirus-transformed host cell is a human embryonic kidney293 cell having DNA encoding the murine dihydrofolatereductase gene, thenascent human protein C gene, the SV40 early promoter, the BK enhancerand the adenovirus 2 late promoter, wherein said SV40 early promoter andsaid adenovirus 2 late promoter are used in tandem.
 12. The method ofclaim 3, further comprising activating the expressed human protein C.